The durability of natural infection and vaccine-induced immunity against future infection by SARS-CoV-2

The durability of vaccine-mediated immunity to SARS-CoV-2, the durations to breakthrough infection, and the optimal timings of booster vaccination are crucial knowledge for pandemic response. Here, we applied comparative evolutionary analyses to estimate the durability of immunity and the likelihood of breakthrough infections over time following vaccination by BNT162b2 (Pfizer-BioNTech), mRNA-1273 (Moderna), ChAdOx1 (Oxford-AstraZeneca), and Ad26.COV2.S (Johnson & Johnson/Janssen). We evaluated anti-Spike (S) immunoglobulin G (IgG) antibody levels elicited by each vaccine relative to natural infection. We estimated typical trajectories of waning and corresponding infection probabilities, providing the distribution of times to breakthrough infection for each vaccine under endemic conditions. Peak antibody levels elicited by messenger RNA (mRNA) vaccines mRNA-1273 and BNT1262b2 exceeded that of natural infection and are expected to typically yield more durable protection against breakthrough infections (median 29.6 mo; 5 to 95% quantiles 10.9 mo to 7.9 y) than natural infection (median 21.5 mo; 5 to 95% quantiles 3.5 mo to 7.1 y). Relative to mRNA-1273 and BNT1262b2, viral vector vaccines ChAdOx1 and Ad26.COV2.S exhibit similar peak anti-S IgG antibody responses to that from natural infection and are projected to yield lower, shorter-term protection against breakthrough infection (median 22.4 mo and 5 to 95% quantiles 4.3 mo to 7.2 y; and median 20.5 mo and 5 to 95% quantiles 2.6 mo to 7.0 y; respectively). These results leverage the tools from evolutionary biology to provide a quantitative basis for otherwise unknown parameters that are fundamental to public health policy decision-making.

The unprecedented development of efficacious vaccines against SARS-CoV-2 has represented a triumph in the global effort to control the ongoing COVID-19 pandemic. Vaccines have been shown to provide short-term protection from major adverse health outcomes of hospitalization and death (1–4). However, protection against breakthrough infection wanes (5), and breakthroughs have been extensively documented (6, 7). In response, the Food and Drug Administration advisory committee has recommended a booster of the Pfizer-BioNTech and Moderna vaccines at least 5 mo after completion of the primary series to people ≥12 and ≥18 y of age, respectively (8). A booster dose of the Johnson & Johnson/Janssen vaccine has been authorized on a faster timescale—as early as 2 mo after the single dose to individuals 18 y of age and older (8). Nevertheless, the optimal timing of boosting remains challenging to assess. Consequently, rigorous prediction of the durability of immunity conferred by vaccination against the SARS-CoV-2 virus is essential to personal and public health decision-making, having major implications regarding policy decisions about COVID-19 vaccination around the world (9, 10).Short-term longitudinal studies of SARS-CoV-2-neutralizing antibodies in vaccinated individuals (11–13) can provide information crucial to our understanding of the durability of vaccine-mediated immunity. Peak antibody responses following vaccination versus natural responses have also been quantified (14), facilitating analytical comparison of initial immune responses. For endemic viruses, longitudinal data on reinfection can provide reinfection probabilities associated with antibody level. However, longitudinal data on SARS-CoV-2 reinfection are not available during the short term associated with pandemic spread. Nevertheless, longitudinal reinfection data for a diversity of coronaviruses have been collected (15–20). SARS-CoV-2 reinfection probabilities have been obtained from them by phylogenetic analysis, using continuous ancestral and descendent state estimation (21). These estimates, produced before reinfection was commonplace, proved accurate (predicting an 18% probability of reinfection at ∼270 d [ref. 21] that was validated by a subsequent empirical finding of 18% reinfection by 275 to 300 d after primary infection [ref. 22] and, likewise, predicting a 34% probability of reinfection at ∼450 d after primary infection [ref. 21] that was validated by a subsequent empirical finding of 34% breakthrough infection 420 to 480 d after primary vaccination [ref. 23]). Similar analyses pairing antibody response and rates of waning for each vaccine with infection probabilities can enable quantification of the durability of vaccine-mediated immunity against breakthrough infections. The aim of this study is to leverage data on antibody response to each vaccine and corresponding probabilities of infection to estimate the durability of vaccine-mediated immunity against breakthrough SARS-CoV-2 infection for four well-studied vaccines: mRNA-1273, BNT162b2, ChAdOx1, and Ad26.COV2.S.     

A single intranasal dose of a live-attenuated parainfluenza virus-vectored SARS-CoV-2 vaccine is protective in hamsters

Single-dose vaccines with the ability to restrict SARS-CoV-2 replication in the respiratory tract are needed for all age groups, aiding efforts toward control of COVID-19. We developed a live intranasal vector vaccine for infants and children against COVID-19 based on replication-competent chimeric bovine/human parainfluenza virus type 3 (B/HPIV3) that express the native (S) or prefusion-stabilized (S-2P) SARS-CoV-2 S spike protein, the major protective and neutralization antigen of SARS-CoV-2. B/HPIV3/S and B/HPIV3/S-2P replicated as efficiently as B/HPIV3 in vitro and stably expressed SARS-CoV-2 S. Prefusion stabilization increased S expression by B/HPIV3 in vitro. In hamsters, a single intranasal dose of B/HPIV3/S-2P induced significantly higher titers compared to B/HPIV3/S of serum SARS-CoV-2–neutralizing antibodies (12-fold higher), serum IgA and IgG to SARS-CoV-2 S protein (5-fold and 13-fold), and IgG to the receptor binding domain (10-fold). Antibodies exhibited broad neutralizing activity against SARS-CoV-2 of lineages A, B.1.1.7, and B.1.351. Four weeks after immunization, hamsters were challenged intranasally with 10^4.5 50% tissue-culture infectious-dose (TCID₅₀) of SARS-CoV-2. In B/HPIV3 empty vector-immunized hamsters, SARS-CoV-2 replicated to mean titers of 10^6.6 TCID₅₀/g in lungs and 10⁷ TCID₅₀/g in nasal tissues and induced moderate weight loss. In B/HPIV3/S-immunized hamsters, SARS-CoV-2 challenge virus was reduced 20-fold in nasal tissues and undetectable in lungs. In B/HPIV3/S-2P–immunized hamsters, infectious challenge virus was undetectable in nasal tissues and lungs; B/HPIV3/S and B/HPIV3/S-2P completely protected against weight loss after SARS-CoV-2 challenge. B/HPIV3/S-2P is a promising vaccine candidate to protect infants and young children against HPIV3 and SARS-CoV-2.

The betacoronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in 2019 and rapidly spread globally (1). In the first year of the pandemic, over 105 million infections and 2.3 million deaths have been reported worldwide, including over 27 million cases and 500,000 deaths in the United States (https://covid19.who.int/). Vaccines are being rapidly deployed in a race to control ongoing infections and the emergence of variants of concern (2), with increased virulence and altered antigenicity.SARS-CoV-2 infects and spreads primarily via the respiratory route (3, 4), and mucosal surfaces of the respiratory tract represent the primary site of infection. COVID-19, the disease caused by SARS-CoV-2, is characterized by upper and lower respiratory tract symptoms, fever, chills, body aches, and fatigue, and in some cases gastrointestinal and other symptoms with involvement of additional tissues (5, 6).SARS-CoV-2 infection is initiated by the spike (S) surface glycoprotein, the main target for SARS-CoV-2–neutralizing antibodies. The S protein is a trimeric class I fusion glycoprotein. Each protomer consists of two functionally distinct subunits, S1 and S2, linked by a furin cleavage site; S2 contains an additional proteolytic cleavage site S2′. S2/S2′ cleavage is mediated by the transmembrane protease serine 2 (TMPRSS2) (1, 7–9). The S1 subunit contains the receptor-binding domain (RBD). The S2 subunit contains the membrane fusion machinery, including the hydrophobic fusion peptide and α-helical heptad repeats (7, 9).Binding of the S RBD to its receptor, human angiotensin converting enzyme 2, triggers a change, from the closed and metastable prefusion conformation to the open and stable postfusion form that drives membrane fusion enabling viral entry (1). Stabilization of the S protein in its native prefusion state should preserve antibody epitopes, including immunodominant sites of the RBD, required to elicit high-quality neutralizing antibody responses (9–13). Thus, a prefusion-stabilized version of the S protein is the optimal vaccine immunogen (13–15).Vaccines for SARS-CoV-2 are available, but currently are limited to individuals 12 y of age or older. They are administered intramuscularly, which does not directly stimulate mucosal immunity in the respiratory tract, the primary site of SARS-CoV-2 infection and shedding. While the major burden of COVID-19 disease is in adults, infection and disease also occurs in infants and young children, contributing to viral transmission. Therefore, the development of safe and effective pediatric COVID-19 vaccines is critical for worldwide control of COVID-19. The ideal vaccine should be effective at a single dose, inducing durable and broad systemic immunity, as well as T and B cell respiratory mucosal immunity that completely blocks SARS-CoV-2 infection and transmission.Here we describe a vectored SARS-CoV-2 vaccine candidate for intranasal immunization of infants and young children. The vaccine is based on an attenuated, replication-competent parainfluenza virus type 3 (PIV3) vector called B/HPIV3 (16) expressing the SARS-CoV-2 S protein. B/HPIV3 consists of bovine PIV3 (BPIV3) strain Kansas in which the BPIV3 hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins (the two PIV3 neutralization antigens) have been replaced by those of human PIV3 strain JS (16, 17). The BPIV3 backbone provides host range restriction of replication in humans, serving as the basis for strong and stable attenuation (17, 18). B/HPIV3 originally was developed as a live vaccine candidate against HPIV3, and was well-tolerated in young children (17). Moreover, B/HPIV3 has been used to express the F glycoprotein of another human respiratory pathogen, human respiratory syncytial virus (HRSV), as a bivalent HPIV3/HRSV vaccine candidate. This vaccine candidate was well-tolerated in children >2 mo of age (18) (Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00686075","term_id":"NCT00686075"}}NCT00686075), and optimized versions are in further clinical development as pediatric vaccines (19, 20). In the present study, we used B/HPIV3 to express wild-type (S) or prefusion-stabilized (S-2P) versions of the SARS-CoV-2 S protein, creating the vaccine candidates B/HPIV3/S and B/HPIV3/S-2P. These were evaluated in vitro and in hamsters as live-attenuated SARS-CoV-2 intranasal vaccine candidates.     

Efficacy and breadth of adjuvanted SARS-CoV-2 receptor-binding domain nanoparticle vaccine in macaques

Emergence of novel variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underscores the need for next-generation vaccines able to elicit broad and durable immunity. Here we report the evaluation of a ferritin nanoparticle vaccine displaying the receptor-binding domain of the SARS-CoV-2 spike protein (RFN) adjuvanted with Army Liposomal Formulation QS-21 (ALFQ). RFN vaccination of macaques using a two-dose regimen resulted in robust, predominantly Th1 CD4+ T cell responses and reciprocal peak mean serum neutralizing antibody titers of 14,000 to 21,000. Rapid control of viral replication was achieved in the upper and lower airways of animals after high-dose SARS-CoV-2 respiratory challenge, with undetectable replication within 4 d in seven of eight animals receiving 50 µg of RFN. Cross-neutralization activity against SARS-CoV-2 variant B.1.351 decreased only approximately twofold relative to WA1/2020. In addition, neutralizing, effector antibody and cellular responses targeted the heterotypic SARS-CoV-1, highlighting the broad immunogenicity of RFN-ALFQ for SARS-CoV−like Sarbecovirus vaccine development.

The COVID-19 pandemic, precipitated by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), continues to threaten global public health and economies. Threats of future outbreaks also loom, as evidenced by three emergent SARS-like diseases caused by zoonotic Betacoronaviruses in the last two decades. While several emergency use authorized (EUA) vaccines currently in use are expected to curb both disease and transmission of SARS-CoV-2 (1–6), the emergence of circulating variants of concern (VOCs) less sensitive to vaccine-elicited immunity has raised concerns for sustained vaccine efficacy (7). Logistical challenges of vaccine production, distribution, storage, and access for these vaccines must be resolved to achieve resolution to the pandemic (8, 9). The rapid and unparalleled spread of SARS-CoV-2 has driven an urgent need to deploy scalable vaccine platforms to combat the ongoing pandemic and mitigate future outbreaks.Current vaccines primarily focus the immune response on the spike glycoprotein (S) as it mediates host cell viral fusion and entry. The receptor-binding domain (RBD) of S engages the primary host cell receptor, angiotensin-converting enzyme 2 (ACE2), for both SARS-CoV-2 and SARS-CoV-1, making RBD a promising domain for vaccine-elicited immune focus (10–12). Moreover, many of the potently neutralizing monoclonal antibodies isolated against SARS-CoV-2 target the RBD (13, 14). Vaccination of nonhuman primates (NHPs) with RBD-encoding RNA or DNA protects against respiratory tract challenge, indicating that immune responses to the RBD can prevent viral replication (15, 16). RBD vaccination also elicits cross-reactive responses to circulating SARS-CoV-2 VOCs in both animals and humans (17, 18), with decrements against the B.1.351 variant similar to that seen with S immunogens (19). The breadth of RBD immunogenicity is further supported by the ability of RBD-specific monoclonal antibodies isolated from SARS-CoV-1 convalescent individuals to cross-neutralize SARS-CoV-2 (20, 21). These findings suggest potential for RBD-based vaccines being efficacious against SARS-CoV-2 variants and other related coronavirus species.Approaches to improve immunogenicity of S or RBD protein vaccines include optimizing antigen presentation and coformulating with adjuvants to enhance the protective immunity. One common approach to enhance the induction of adaptive immune responses is the multimeric presentation of antigen, for example, on the surface of nanoparticles or virus-like particles (22). Presenting RBD in ordered, multivalent arrays on the surface of self-assembling protein nanoparticles is immunogenic and efficacious in animals (23–28), with improved immunogenicity relative to monomeric soluble RBD and cross-reactive responses to variants (17, 24, 26). However, it is unknown whether RBD nanoparticle vaccines protect against infection in primates, which have become a standard model for benchmarking performance of vaccine candidates by virological and immunologic endpoints. Liposomal adjuvants incorporating QS-21, such as that used in the efficacious varicella zoster vaccine, SHINGRIX, may augment protective immunity to SARS-CoV-2 vaccines. Such adjuvants have superior humoral and cellular immunogenicity relative to conventional adjuvants (29, 30).Here, we evaluate the use of a ferritin nanoparticle vaccine presenting the SARS-CoV-2 RBD (RFN) adjuvanted with the Army Liposomal Formulation QS-21 (ALFQ) (31). Both ferritin nanoparticles and ALFQ have been evaluated for vaccination against multiple pathogens in humans in phase 1 clinical trials (32–34). We demonstrate, in an NHP model, that immunization with RFN induces robust and broad antibody and T cell responses, as well as protection against viral replication and lung pathology following high-dose respiratory tract challenge with SARS-CoV-2.     

Heterologous vaccination interventions to reduce pandemic morbidity and mortality: Modeling the US winter 2020 COVID-19 wave

COVID-19 remains a stark health threat worldwide, in part because of minimal levels of targeted vaccination outside high-income countries and highly transmissible variants causing infection in vaccinated individuals. Decades of theoretical and experimental data suggest that nonspecific effects of non–COVID-19 vaccines may help bolster population immunological resilience to new pathogens. These routine vaccinations can stimulate heterologous cross-protective effects, which modulate nontargeted infections. For example, immunization with Bacillus Calmette–Guérin, inactivated influenza vaccine, oral polio vaccine, and other vaccines have been associated with some protection from SARS-CoV-2 infection and amelioration of COVID-19 disease. If heterologous vaccine interventions (HVIs) are to be seriously considered by policy makers as bridging or boosting interventions in pandemic settings to augment nonpharmaceutical interventions and specific vaccination efforts, evidence is needed to determine their optimal implementation. Using the COVID-19 International Modeling Consortium mathematical model, we show that logistically realistic HVIs with low (5 to 15%) effectiveness could have reduced COVID-19 cases, hospitalization, and mortality in the United States fall/winter 2020 wave. Similar to other mass drug administration campaigns (e.g., for malaria), HVI impact is highly dependent on both age targeting and intervention timing in relation to incidence, with maximal benefit accruing from implementation across the widest age cohort when the pandemic reproduction number is >1.0. Optimal HVI logistics therefore differ from optimal rollout parameters for specific COVID-19 immunizations. These results may be generalizable beyond COVID-19 and the US to indicate how even minimally effective heterologous immunization campaigns could reduce the burden of future viral pandemics.

On March 16th, 2020, Imperial College London released a landmark report advocating the suppression of SARS-CoV-2 to avoid a pandemic catastrophe (1). Since then, the scientific community has been challenged to create a “bridge period” of reduced COVID-19 morbidity and mortality until safe and effective targeted vaccines are delivered globally (2). Guided by major modeling groups and international and national public health authorities, most countries quickly implemented variably stringent nonpharmaceutical interventions (NPIs) including physical distancing, self-isolation, home working, school closure, and “shielding” of vulnerable populations such as the elderly. Despite ameliorating COVID-19 incidence when applied, these “lockdowns” of regional and national economies also caused severe financial, social, and health repercussions globally (3). In the United States and other countries, resistance to and reversal of NPIs occurred in many jurisdictions, complicating pandemic control and contributing to persistently high COVID-19 incidence.The rollout of specific COVID-19 vaccines in 2021 led to a temporary reduction of pandemic caseloads in countries with effective vaccine campaigns and ample stocks, but even this has not proven to be the sought-for panacea for epidemiological, logistical, and political reasons. The emergence of virus variants—now dominated by the Omicron and Delta strains—that are more transmissible and pathogenic have reversed many gains achieved to date and have raised questions about the durability of current vaccine efficacy (4). Although a handful of mainly high-income countries have instituted vigorous campaigns that have rapidly provided high coverage, less than 5% of the world’s low-income population has received at least one COVID-19–specific vaccination (5), and even in countries with ample vaccine supply, the global phenomenon of multifactorial vaccine hesitancy has led to uneven intranational uptake that has been exploited by the Delta variant. For these reasons, the public health armamentarium against COVID-19 has ample room for adjuncts to both NPIs and COVID-19–specific vaccines.One as-yet unutilized intervention to potentially prevent SARS-CoV-2 infection and reduce COVID-19 disease is based upon heterologous or nonspecific effects (NSEs) induced by available non–SARS-CoV-2 vaccines (6). The heterologous effect of vaccination refers to the impact that vaccines can have on unrelated infections and diseases. These effects have been noted for almost a hundred years (7), and potential mechanisms include innate and adaptive immune responses. Trained immunity (8–14), increased cytokine production (15–17), viral interference (18), long-lasting type I interferons (19), the antiviral state (20), cross-reactivity (21, 22), and bystander activation (23) are some of the mechanisms proposed.Some of the best-studied heterologous vaccine actions are from “off-target effects” from the Bacillus Calmette–Guérin (BCG) vaccine (12, 24–29). Epidemiological evidence including several randomized controlled trials (RCTs) have assessed the effect of BCG vaccination on reducing neonatal mortality. In Guinea-Bissau, two RCTs of BCG given to low weight neonates showed reduction in neonatal mortality after BCG, mainly because of fewer cases of neonatal sepsis, respiratory infection, and fever (30, 31). A meta-analysis of three RCTs of BCG-Denmark showed a reduction in mortality rate of 38% at 28 d of life; marked reductions in mortality were also seen within 3 d after vaccination and at 12 mo of age (32). Interestingly, a BCG vaccination prior to an influenza vaccination can boost influenza-specific immunity (33).Because of the nonspecific benefits of BCG vaccination, a phase III trial called “ACTIVATE-2” assessed whether BCG could protect against COVID-19 in the elderly; prepublication findings suggest a 68% risk reduction for total COVID-19 clinical and microbiological diagnoses (34). A separate study showed that a history of BCG vaccination was associated with a decreased SARS-CoV-2 seroprevalence across a diverse cohort of healthcare workers, and reduced COVID-19 symptoms (35). The magnitude of protective effect against symptomatic disease was similar in both studies: a reported range of 10 to 30% reduction in all respiratory infections in the former and a 34.5% reduction in self-reported diagnosis of COVID-19 in the latter.Other epidemiological studies have shown NSE benefits from oral polio vaccine (OPV), measles-containing vaccines (MCVs), and several other common immunizations. OPV has been associated with beneficial NSE (20, 36–38) and may become pronounced with subsequent doses (39–41). A systematic review of the associations of BCG, diptheria-tetanus-pertussis, and MCVs with childhood mortality showed that BCG and MCVs reduced overall mortality by more than would be expected through their effects on the diseases they target (42). As with BCG, an RCT of MCV showed a beneficial nonspecific effect on children’s survival (43).Focusing on SARS-CoV-2 transmission, several studies have found that the administration of OPV, Hemophilus influenza type-B, measles mumps rubella (MMR), varicella, hepatitis A/B, pneumococcal conjugate, and inactivated influenza vaccines are associated with decreased SARS-CoV-2 infection rates (44–46). In addition, results from a study in a Dutch hospital showed a 37 to 49% lower risk of SARS-CoV-2 infection in healthcare workers who received the influenza vaccine in the previous flu season, and this finding was also corroborated by a preliminary in vitro study (9). Thus, there is some evidence to support an impact of routine vaccinations on SARS-CoV-2 infection rates, although these ecological studies are prone to bias, do not establish causality, and may be SARS-CoV-2 variant-specific.Vaccine-mediated heterologous effects could also extend to reducing the severity of COVID-19 disease. There are epidemiological associations between those who have had a past vaccination with BCG, MMR, inactivated influenza vaccine, and recombinant adjuvanted zoster vaccine and reduced mortality and/or reduced COVID-19 severity (35, 45, 47–57), although these additional ecological studies are similarly susceptible to bias. A recent interim analysis of an ongoing clinical trial in Brazil supports this claim, showing that vaccination with MMR reduces the risk of COVID-19 symptoms and need for treatment (58). Given that the COVID-19 pandemic is still a global health emergency (especially in undervaccinated countries) and that the premise of HVI is soundly based in the immunological and epidemiological literature, there is ample reason to consider its potential role as part of a comprehensive package of pandemic control strategies.The plethora of studies cited can help characterize the hypothetical efficacy of immune system boosting through HVIs to reduce COVID-19 morbidity and mortality. However, estimating the potential real-world effectiveness of such interventions requires their implementation in an environment that can quantify their potential population-level impact in the context of ongoing control measures on viral transmission, health care utilization, and health outcomes. This type of epidemiological projection can be achieved through the use of mathematical models of infectious disease (59–65).We used the COVID-19 International Modeling (CoMo) Consortium Model (https://comomodel.net), an open-source, age-structured, country-specific, dynamic compartmental model of SARS-CoV-2 transmission and COVID-19 illness, treatment, and mortality, to illustrate how the logistics of implementing a heterologous vaccine intervention (HVI)—in particular, the timing of initiation of such a vaccination campaign in relation to trends in disease incidence and also the age-related population targeting of such a campaign—largely determine the magnitude of their impact. In particular, we instituted an explicitly defined HVI in one of three distinct time frames during the large fall/winter wave of SARS-CoV-2 in the United States (presurge, intrasurge, and postsurge) and across the same total number of individuals in one of three distinct age-targeted population groups (20+ y old, 40+ y old, and 65+ y old).There are multiple potential applications of heterologous vaccination in this setting, e.g., as a pre–COVID-19 vaccination primer, as a simultaneously delivered or post–COVID-19 vaccination booster (i.e., replacing or delaying the use of a second COVID-specific vaccine dose), or as a solitary “bridging” intervention to reduce or delay COVID-19-related morbidity and mortality until a specific vaccine is available. Here, we explore the last use: that of a solo heterologous vaccination used as a temporizing “bridging” intervention that has only a low level of heterologous effectiveness at reducing viral transmission (here defined as reducing the likelihood of being infected by 5, 10, or 15%) and clinical severity (i.e., reducing the risk of death if infected, again by 5, 10, or 15%). Given the high levels of targeted vaccination now attained in many high-income countries, our results with respect to the prevaccinated US outbreak should be seen as general, model-informed operational guidance that could maximize the beneficial effect of efforts to use common vaccination programs to mitigate and temporize the impact of COVID-19, and possibly future viral pandemics, in the majority of countries worldwide that have not yet received sufficient quantities of COVID-19–specific vaccines to ensure population protection.     

Targeted vaccination and the speed of SARS-CoV-2 adaptation

The limited supply of vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) raises the question of targeted vaccination. Many countries have opted to vaccinate older and more sensitive hosts first to minimize the disease burden. However, what are the evolutionary consequences of targeted vaccination? We clarify the consequences of different vaccination strategies through the analysis of the speed of viral adaptation measured as the rate of change of the frequency of a vaccine-adapted variant. We show that such a variant is expected to spread faster if vaccination targets individuals who are likely to be involved in a higher number of contacts. We also discuss the pros and cons of dose-sparing strategies. Because delaying the second dose increases the proportion of the population vaccinated with a single dose, this strategy can both speed up the spread of the vaccine-adapted variant and reduce the cumulative number of deaths. Hence, strategies that are most effective at slowing viral adaptation may not always be epidemiologically optimal. A careful assessment of both the epidemiological and evolutionary consequences of alternative vaccination strategies is required to determine which individuals should be vaccinated first.

The development of effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) raises hope regarding the possibility of eventually halting the ongoing pandemic. However, vaccine supply shortages have sparked a debate about the optimal distribution of vaccination among different categories of individuals. Typically, infections with SARS-CoV-2 are far more deadly in older individuals than in younger ones (1). Prioritizing vaccination for older classes may thus provide a direct benefit in terms of mortality (2, 3). Yet, younger individuals are usually more active, and consequently, they may contribute more to the spread of the epidemic. Prioritizing vaccination for younger and more active individuals may thus provide an indirect benefit through a reduction of the epidemic size (4, 5). Earlier studies have compared alternative ways to deploy vaccination in heterogeneous host populations and showed that recommendation varies with the choice of the quantity one is trying to minimize (e.g., the cumulative number of deaths, the remaining life expectancy, or the number of infections) (3, 6, 7). The recommendation also varies with the properties of the pathogen and the efficacy of the vaccine (3, 4, 8). For SARS-CoV-2, the increase in mortality with age is such that the direct benefit associated with vaccinating more vulnerable individuals tends to overwhelm the indirect benefits obtained from vaccinating more active individuals (2, 3, 9, 10). However, some studies challenge this view and identified specific conditions where vaccinating younger and more active classes could be optimal (5, 7, 11, 12). A similar debate emerges over the possibility to delay the second vaccination dose to maximize the number of partially vaccinated individuals. A quantitative exploration of alternative vaccination strategies can help provide useful recommendations: a two-dose strategy is recommended when the level of protection obtained after the first dose is low and/or when vaccine supply is large (13–16).Vaccine-driven evolution, however, could erode the benefit of vaccination and alter the above recommendations which are based solely on the analysis of epidemiological dynamics. Given that hosts differ both in their sensitivity to the disease and in their contribution to transmission, who should we vaccinate first if we want to minimize the spread of vaccine-adapted variants? The effect of alternative vaccination strategies on the speed of pathogen adaptation remains unclear. Previous studies of adaptation to vaccines focused on long-term evolutionary outcomes (17, 18). These analyses are not entirely relevant for the ongoing pandemic because what we want to understand first is the short-term consequence of different vaccination strategies (19). A few studies have discussed the possibility of SARS-CoV-2 adaptation following different targeted vaccination strategies but did not explicitly account for evolutionary dynamics (12, 20). A recent simulation study explored the effect of a combination of vaccination and social distancing strategies on the probability of vaccine-driven adaptation (21). This model, however, did not study the impact of targeted vaccination strategies on the speed of adaptation.Here we develop a theoretical framework based on the analysis of the deterministic dynamics of multiple variants after they successfully managed to reach a density at which they are no longer affected by the action of demographic stochasticity. We study the impact of different vaccination strategies on the rate of change of the frequency of a novel variant, which allows us to quantify the speed of virus adaptation to vaccines. Numerical simulations tailored to the epidemiology of SARS-CoV-2 confirm the validity of our approximation of the strength of selection for vaccine-adapted variants.     

Early human B cell signatures of the primary antibody response to mRNA vaccination

Messenger RNA (mRNA) vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are highly effective at inducing protective immunity. However, weak antibody responses are seen in some individuals, and cellular correlates of immunity remain poorly defined, especially for B cells. Here we used unbiased approaches to longitudinally dissect primary antibody, plasmablast, and memory B cell (MBC) responses to the two-dose mRNA-1273 vaccine in SARS-CoV-2–naive adults. Coordinated immunoglobulin A (IgA) and IgG antibody responses were preceded by bursts of spike-specific plasmablasts after both doses but earlier and more intensely after dose 2. While antibody and B cell cellular responses were generally robust, they also varied within the cohort and decreased over time after a dose-2 peak. Both antigen-nonspecific postvaccination plasmablast frequency after dose 1 and their spike-specific counterparts early after dose 2 correlated with subsequent antibody levels. This correlation between early plasmablasts and antibodies remained for titers measured at 6 months after vaccination. Several distinct antigen-specific MBC populations emerged postvaccination with varying kinetics, including two MBC populations that correlated with 2- and 6-month antibody titers. Both were IgG-expressing MBCs: one less mature, appearing as a correlate after the first dose, while the other MBC correlate showed a more mature and resting phenotype, emerging as a correlate later after dose 2. This latter MBC was also a major contributor to the sustained spike-specific MBC response observed at month 6. Thus, these plasmablasts and MBCs that emerged after both the first and second doses with distinct kinetics are potential determinants of the magnitude and durability of antibodies in response to mRNA-based vaccination.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) instigated rapid worldwide COVID-19 vaccine prioritization strategies. Several vaccine candidates were developed, including two vaccines (Moderna mRNA-1273 and the Pfizer/BioNTech BNT162b2), based on novel messenger RNA (mRNA) platforms (1). Both mRNA vaccines encode a stabilized ectodomain of the spike protein trimer (S-2P) derived from the Wuhan Hu-1 isolate (2). Two doses of mRNA vaccines have been shown to be highly protective and elicit strong antibody responses (3, 4), although poorer responses have also been seen in some individuals, such as older adults (5, 6) and transplant recipients (7, 8), raising the question of what determines antibody response levels and whether cellular correlates can be defined. Several studies have shown that SARS-CoV-2 mRNA vaccines can elicit a durable cellular response, including among B cells (reviewed in (9)), with memory B cells (MBCs) shown to correlate with the antibody response (10).In the B cell compartment, one of the first detected responses in the blood after a primary immunization is a short transient burst around days 7–10 of plasmablasts (PBs) that are probably induced by the extrafollicular response and potentially responsible for the early serum antibodies to the immunogen, reviewed in (11). While it is unclear whether PBs are direct precursors of bone marrow–resident plasma cells that are the main source of circulating antibodies (12), several studies on inactivated and attenuated vaccines have shown that the PB response can predict the magnitude and longevity of protective antibodies (13–16). Among these predictors are PB responses that are independent of antigen specificity (17, 18), suggesting that the quantitative extent of antigen-specific responses is coupled to that of the total PB responses detectable in blood, including bystander and PBs with weak affinity for detection (13, 15). For mRNA-based SARS-CoV-2 vaccines, several studies have described a robust yet highly variable PB response in blood and draining lymph nodes (5, 19), and there is evidence of a clonal relationship between PBs in the blood and MBCs in the lymph nodes (20). Despite these advances, the role of PBs and other B cell populations in the induction and longevity of antibodies following mRNA-based vaccination and how they differ across individuals and potentially contribute to variability in antibody responses have not been fully assessed.Here we performed parallel antibody and cellular assays on frequent blood collections to capture the early events of the primary B cell response to the mRNA vaccine mRNA-1273. Using an unbiased approach, we identified PBs and other early B cell populations as correlates of the antibody response to SARS-CoV-2 mRNA-based vaccination.     

Immunogenicity and efficacy of the COVID-19 candidate vector vaccine MVA-SARS-2-S in preclinical vaccination

Severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) has emerged as the infectious agent causing the pandemic coronavirus disease 2019 (COVID-19) with dramatic consequences for global human health and economics. Previously, we reached clinical evaluation with our vector vaccine based on modified vaccinia virus Ankara (MVA) against the Middle East respiratory syndrome coronavirus (MERS-CoV), which causes an infection in humans similar to SARS and COVID-19. Here, we describe the construction and preclinical characterization of a recombinant MVA expressing full-length SARS-CoV-2 spike (S) protein (MVA-SARS-2-S). Genetic stability and growth characteristics of MVA-SARS-2-S, plus its robust expression of S protein as antigen, make it a suitable candidate vaccine for industrial-scale production. Vaccinated mice produced S-specific CD8⁺ T cells and serum antibodies binding to S protein that neutralized SARS-CoV-2. Prime-boost vaccination with MVA-SARS-2-S protected mice sensitized with a human ACE2-expressing adenovirus from SARS-CoV-2 infection. MVA-SARS-2-S is currently being investigated in a phase I clinical trial as aspirant for developing a safe and efficacious vaccine against COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of coronavirus disease 2019 (COVID-19), first emerged in late 2019 in China (1). SARS-CoV-2 exhibits extremely efficient human-to-human transmission, the new pathogen rapidly spread worldwide, and within months it caused a global pandemic, changing daily life for billions of people. The COVID-19 case fatality rate of ∼2–5% makes the development of countermeasures a global priority. In fact, the development of COVID-19 vaccine candidates is advancing at an international level with unprecedented speed. About 1 y after the first known cases of COVID-19, we can account for >80 SARS-CoV-2–specific vaccines in clinical evaluations and >10 candidate vaccines already in phase III trials (2–4). However, we still lack information on the key immune mechanisms needed for protection against COVID-19. A better understanding of the types of immune response elicited upon natural SARS-CoV-2 infections has become an essential component to assess the promise of various vaccination strategies (5).The SARS-CoV-2 spike (S) protein serves as the most important target antigen for vaccine development based on preclinical research on candidate vaccines against SARS-CoV or Middle East respiratory syndrome coronavirus (MERS-CoV). The trimeric S protein is a prominent structure at the virion surface and essential for SARS-CoV-2 cell entry. As a class I viral fusion protein, it mediates virus interaction with the cellular receptor angiotensin-converting enzyme 2 (ACE2), and fusion with the host cell membrane, both key steps in infection. Thus, infection can be prevented by S-specific antibodies neutralizing the virus (6–9).Among the front-runner vaccines are new technologies such as messenger RNA (mRNA)-based vaccines and nonreplicating adenovirus vector vaccines (10–13). First reports from these SARS-CoV-2-S–specific vaccines in phase 1/2 clinical studies demonstrated acceptable safety and promising immunogenicity profiles, and by now data from large phase 3 clinical trials show promising levels of protective efficacy (4, 12–14). In December 2020, the first mRNA-based COVID-19 vaccines received emergency use authorization or conditional licensing by the US Food and Drug Administration and European Medicines Agency (11, 15, 16). By March 2021, two adenovirus vector-based COVID-10 vaccines had been approved by regulatory authorities (17, 18). This is good news because efficacious vaccines will provide a strategy to change SARS-CoV-2 transmission dynamics. In addition, multiple vaccine types will be advantageous to meet specific demands across different target populations. This includes the possibility of using heterologous immunization strategies depending on an individual’s health status, boosting capacities, and the need for balanced humoral and Th1-directed cellular immune responses.MVA, a highly attenuated strain of vaccinia virus originating from growth selection on chicken embryo tissue cultures, shows a characteristic replication defect in mammalian cells but allows unimpaired production of heterologous proteins (19). At present, MVA serves as an advanced vaccine technology platform for developing new vector vaccines against infectious disease including emerging viruses and cancer (20). In response to the ongoing pandemic, the MVA vector vaccine platform allows rapid generation of experimental SARS-CoV-2–specific vaccines (21). Previous work from our laboratory addressed the development of an MVA candidate vaccine against MERS with immunizations in animal models demonstrating the safety, immunogenicity, and protective efficacy of MVA-induced MERS-CoV S-antigen–specific immunity (22–25). Clinical safety and immunogenicity of the MVA-MERS-S candidate vaccine was established in a first-in-human phase I clinical study under funding from the German Center for Infection Research (DZIF) (26).Here, we show that a recombinant MVA produces the full-length S protein of SARS-CoV-2 as ∼190- to 200-kDa N-glycosylated protein. Our studies confirmed cleavage of the mature full-length S protein into an amino-terminal domain (S1) and a ∼80- to 100-kDa carboxyl-terminal domain (S2) that is anchored to the membrane. When tested as a vaccine in BALB/c mice, recombinant MVA expressing the S protein induced SARS-CoV-2–specific T cells and antibodies, and robustly protected vaccinated animals against lung infection upon SARS-CoV-2 challenge.     

How immunity from and interaction with seasonal coronaviruses can shape SARS-CoV-2 epidemiology

We hypothesized that cross-protection from seasonal epidemics of human coronaviruses (HCoVs) could have affected severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission, including generating reduced susceptibility in children. To determine what the prepandemic distribution of immunity to HCoVs was, we fitted a mathematical model to 6 y of seasonal coronavirus surveillance data from England and Wales. We estimated a duration of immunity to seasonal HCoVs of 7.8 y (95% CI 6.3 to 8.1) and show that, while cross-protection between HCoV and SARS-CoV-2 may contribute to the age distribution, it is insufficient to explain the age pattern of SARS-CoV-2 infections in the first wave of the pandemic in England and Wales. Projections from our model illustrate how different strengths of cross-protection between circulating coronaviruses could determine the frequency and magnitude of SARS-CoV-2 epidemics over the coming decade, as well as the potential impact of cross-protection on future seasonal coronavirus transmission.

Due to the relatively short time since severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged, little is yet known about the duration of infection-induced immunity. While instances of confirmed reinfection of SARS-CoV-2 have been identified (1), these are rare (2), indicating protection lasts for at least 6 mo to 8 mo, which concurs with estimates from prospective studies (3, 4). Cross-protection from seasonal human coronaviruses (HCoVs) could have impacted the transmission dynamics of SARS-CoV-2, and explain the relatively low SARS-CoV-2 infection rate in children (5–8). Since children likely have a higher annual attack rate of endemic HCoVs due to their higher contact rates (9), they may be less susceptible to SARS-CoV-2 due to cross-protection.In order to evaluate the impacts of cross-immunity, we first need to quantify the immune protection from seasonal coronaviruses. Four coronavirus strains from two different genera are endemic in humans: Two are alphacoronaviruses (HCoV-229E and HCoV-NL63), and two are betacoronaviruses (HCoV-HKU1 and HCoV-OC43); SARS-CoV-2 is a member of the latter genera, as are SARS-CoV-1 and Middle East respiratory syndrome coronavirus (MERS-CoV). In the United Kingdom, seasonal human coronavirus (HCoV) case incidence peaks January–February each year. The first infection with seasonal HCoVs typically occurs in childhood (10), and reinfection with the same strain has been observed within a year (11, 12). However, there are also indications that immunity lasts longer, with few reinfections in a 3-y cohort study (13) and sterilizing immunity to homologous strains of HCoV-229E after 1 y in a challenge study (14).There may also be cross-protective immunity between seasonal HCoVs and SARS-family coronaviruses following infection. Human sera collected before the SARS-CoV-2 pandemic showed high IgG reactivity to seasonal HCoVs, but also low reactivity to SARS-CoV-2 (15), and SARS-CoV-1 infection induced antibody production against seasonal HCoVs (16, 17). Cross-reactive T cells to SARS-CoV-2 have been found in 20% (18) to 50% (38) of unexposed individuals, with evidence that these responses stem from seasonal coronavirus infection (20). It has also been noted that these are more prevalent in children and adolescents (21).Cross-protection from seasonal HCoVs may have, therefore, partially shaped the observed epidemiology of SARS-CoV-2. Using England and Wales as a case study, we use dynamic models to estimate 1) the duration of infection-induced immunity to seasonal HCoVs, 2) the ability of potential cross-protection from seasonal HCoVs to explain the age patterns in the first wave of the SARS-CoV-2 pandemic, and 3) the implications of the duration of immunity and potential cross-protection on future dynamics of SARS-CoV-2.     

Interferon resistance of emerging SARS-CoV-2 variants

The emergence of SARS-CoV-2 variants with enhanced transmissibility, pathogenesis, and resistance to vaccines presents urgent challenges for curbing the COVID-19 pandemic. While Spike mutations that enhance virus infectivity or neutralizing antibody evasion may drive the emergence of these novel variants, studies documenting a critical role for interferon responses in the early control of SARS-CoV-2 infection, combined with the presence of viral genes that limit these responses, suggest that interferons may also influence SARS-CoV-2 evolution. Here, we compared the potency of 17 different human interferons against multiple viral lineages sampled during the course of the global outbreak, including ancestral and five major variants of concern that include the B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma), B.1.617.2 (delta), and B.1.1.529 (omicron) lineages. Our data reveal that relative to ancestral isolates, SARS-CoV-2 variants of concern exhibited increased interferon resistance, suggesting that evasion of innate immunity may be a significant, ongoing driving force for SARS-CoV-2 evolution. These findings have implications for the increased transmissibility and/or lethality of emerging variants and highlight the interferon subtypes that may be most successful in the treatment of early infections.

The human genome encodes a diverse array of antiviral interferons (IFNs). These include the type I IFNs (IFN-Is) such as the 12 IFNα subtypes IFNβ and IFNω that signal through the ubiquitous IFNΑR (IFN α-receptor), and the type III IFNs (IFN-IIIs) such as IFNλ1, IFNλ2, and IFNλ3 that signal through the more restricted IFNλR receptor that is present in lung epithelial cells (1). IFN diversity may be driven by an evolutionary arms race in which viral pathogens and hosts reciprocally evolve countermeasures (2). For instance, the IFNα subtypes exhibit >78% amino acid sequence identity, but IFNα14, IFNα8, and IFNα6 most potently inhibited HIV-1 in vitro and in vivo (3–5), whereas IFNα5 most potently inhibited influenza H3N2 in lung explant cultures (6). Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) was sensitive to IFNα2, IFNβ, and IFNλ (7–9), and several clinical trials of IFNα2 and IFNβ demonstrated therapeutic promise for coronavirus disease 2019 (COVID-19) (10–12). A recent Phase III clinical trial demonstrating that IFNβ may not have therapeutic benefits in vivo (13) could be due to the late timing of administration among hospitalized/severely ill patients, cotreatment with glucocorticoids such as dexamethasone that directly antagonize IFN signaling (14), and the use of a subcutaneous route that may not efficiently target IFNs to pulmonary epithelial cells. To date, a direct comparison of multiple IFN-Is and IFN-IIIs against diverse SARS-CoV-2 variants of concern has not yet been done.The present study was initiated to determine which IFNs would best inhibit SARS-CoV-2. Initially, we selected five isolates from prominent lineages (15) during the early phase of the pandemic (Fig. 1 and SI Appendix, Table S1). USA-WA1/2020 is the standard strain used in many in vitro and in vivo studies of SARS-CoV-2 and belongs to lineage A (15). It was isolated from the first COVID-19 patient in the United States, who had a direct epidemiologic link to Wuhan, China, where the virus was initially detected (16). By contrast, subsequent infection waves from Asia to Europe (17) were associated with the emergence of the D614G mutation (18). Lineage B strains with G614 spread globally and displaced ancestral viruses with striking speed, likely due to increased transmissibility (19, 20). These strains accumulated additional mutations in Italy as lineage B.1, which then precipitated a severe outbreak in New York City (21). Later, in the United Kingdom, lineage B.1.1.7 acquired an N501Y mutation associated with enhanced transmissibility (15). Lineage B.1.351, first reported in South Africa, additionally acquired an additional E484K mutation associated with resistance to neutralizing antibodies (22, 23). Both B.1.1.7 and B.1.351 were reported in multiple countries, and in some cases have become dominant for extended periods (24). Subsequent waves of infection after our initial preprint was deposited in March 2021 (25) was associated with the P.1, B.1.617.2, and B.1.1.529 lineages (26–29). The emergence of these novel variants provided a unique opportunity to determine whether SARS-CoV-2 has evolved since its initial introduction into humans to better counteract innate immune selection pressures driven by the antiviral IFNs.Open in a separate windowFig. 1.Selection of SARS-CoV-2 strains for IFN sensitivity studies. (A) Global distribution of SARS-CoV-2 clades. GISAID.org plotted the proportion of deposited sequences in designated clades against collection dates. The 10 isolates chosen are noted by colored dots. (B) SARS-CoV-2 strains selected for this study included representatives of lineages A, B, B.1, B.1.351, and B.1.1.7 (SI Appendix, Table S1). Lineage P.1 (which branched off from lineage B.1.1.28), B.1.617.2, and B.1.1.529 were added after the initial preprint submission and were evaluated for IFNβ and IFNλ1 sensitivity. Lineage B isolates encode the D614G mutation associated with increased transmissibility. *Amino acid mutations were relative to the reference hCOV-19/Wuhan/WIV04/2019 sequence.     

Vaccine-induced systemic and mucosal T cell immunity to SARS-CoV-2 viral variants

The first-generation COVID-19 vaccines have been effective in mitigating severe illness and hospitalization, but recurring waves of infections are associated with the emergence of SARS-CoV-2 variants that display progressive abilities to evade antibodies, leading to diminished vaccine effectiveness. The lack of clarity on the extent to which vaccine-elicited mucosal or systemic memory T cells protect against such antibody-evasive SARS-CoV-2 variants remains a critical knowledge gap in our quest for broadly protective vaccines. Using adjuvanted spike protein–based vaccines that elicit potent T cell responses, we assessed whether systemic or lung-resident CD4 and CD8 T cells protected against SARS-CoV-2 variants in the presence or absence of virus-neutralizing antibodies. We found that 1) mucosal or parenteral immunization led to effective viral control and protected against lung pathology with or without neutralizing antibodies, 2) protection afforded by mucosal memory CD8 T cells was largely redundant in the presence of antibodies that effectively neutralized the challenge virus, and 3) “unhelped” mucosal memory CD8 T cells provided no protection against the homologous SARS-CoV-2 without CD4 T cells and neutralizing antibodies. Significantly, however, in the absence of detectable virus-neutralizing antibodies, systemic or lung-resident memory CD4 and “helped” CD8 T cells provided effective protection against the relatively antibody-resistant B1.351 (β) variant, without lung immunopathology. Thus, induction of systemic and mucosal memory T cells directed against conserved epitopes might be an effective strategy to protect against SARS-CoV-2 variants that evade neutralizing antibodies. Mechanistic insights from this work have significant implications in the development of T cell–targeted immunomodulation or broadly protective SARS-CoV-2 vaccines.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continued to exert devastating impacts on the human life, with >280 million infections and over 5.4 million deaths to date. Although there are millions of convalescent people with some measure of immunity and 8.8 billion doses of vaccine administered to date, further threats of widespread severe COVID-19 disease looms heavily as immunity induced by infection or the first-generation vaccines may not provide effective and durable protection, either due to waning immunity or due to poor antibody cross-reactivity to new variants (1–5).It is clear that virus-neutralizing antibodies provide the most effective protection to SARS-CoV-2, following vaccination or recovery from infection (6). However, T cell–based protection against SARS-CoV-2 has become a central focus because T cells recognize short amino acid sequences that can be conserved across viral variants (7–9). Indeed, T cells in convalescent COVID-19 patients have shown robust responses that are directed at multiple viral proteins, and depletion of these T cells delayed SARS-CoV-2 control in mice (10–12). These data suggest a protective role for T cells in COVID-19 infection. In effect, what constitutes an effective, an ineffective, or a perilous T cell response to SARS-CoV-2 in lungs remains poorly defined. Controlled studies in laboratory animals are of critical importance to elucidate the role and nature of T cells in lungs during SARS-CoV-2 virus infection and in protective immunity.Based on the differentiation state, anatomical localization and traffic patterns, memory T cells are classified into effector memory (T_EM), central memory (T_CM), and tissue-resident memory (T_RM) (13, 14). There is accumulating evidence that airway/lung-resident T_RMs, and not migratory memory T cells (T_EMs) are critical for protective immunity to respiratory mucosal infections with viruses, such as influenza A virus (IAV) and respiratory syncytial virus (15–21). Development of T_RMs from effector T cells in the respiratory tract requires local antigen recognition and exposure to critical factors, such as transforming growth factor (TGF)-β and interleukin (IL)-15 (15). Therefore, mucosal vaccines are more likely to elicit T_RMs in lungs than parenteral vaccines (22, 23). A subset of effector T cells in airways of COVID-19 patients display T_RM-like features (24), but the development of T_RMs or their importance in protective immunity to reinfection are yet to be determined. Furthermore, all SARS-CoV-2 vaccines in use are administered parenterally and less likely to induce lung T_RMs. While depletion of CD8 T cells compromised protection against COVID-19 in vaccinated rhesus macaques (25), the relative effectiveness of vaccine-induced systemic/migratory CD8 T cell memory vs. lung/airway T_RMs in protective immunity to COVID-19 is yet to be defined.In this study, using the K18-hACE2 transgenic (tg) mouse model of SARS-CoV-2 infection, we have interrogated two key aspects of T cell immunity: 1) the requirements for lung-resident vs. migratory T cell memory in vaccine-induced immunity to SARS-CoV-2; and 2) the role of lung-resident memory CD4 vs. CD8 T cells in protection against viral variants in the presence or absence of virus-neutralizing antibodies. Studies of mucosal versus systemic T cell–based vaccine immunity using a subunit protein-based adjuvant system that elicits neutralizing antibodies and T cell immunity, demonstrated that: 1) both mucosal and parenteral vaccinations provide effective immunity to SARS-CoV-2 variants; 2) CD4 T cell–dependent immune mechanisms exert primacy in protection against homologous SARS-CoV-2 strain; and 3) the development of spike (S) protein-specific “unhelped” memory CD8 T cells in the respiratory mucosa are insufficient to protect against a lethal challenge with the homologous Washington (WA) strain of SARS-CoV-2. Unexpectedly, we found that systemic or mucosal lung-resident memory CD4 and “helped” CD8 T cells engendered effective immunity to the South African B1.351 β-variant in the apparent absence of detectable mucosal or circulating virus-neutralizing antibodies. Taken together, mechanistic insights from this study have advanced our understanding of viral pathogenesis and might drive rational development of next-generation broadly protective SARS-CoV-2 vaccines that induce humoral and T cell memory.     

SARS-CoV-2 evolution in animals suggests mechanisms for rapid variant selection

SARS-CoV-2 spillback from humans into domestic and wild animals has been well documented, and an accumulating number of studies illustrate that human-to-animal transmission is widespread in cats, mink, deer, and other species. Experimental inoculations of cats, mink, and ferrets have perpetuated transmission cycles. We sequenced full genomes of Vero cell–expanded SARS-CoV-2 inoculum and viruses recovered from cats (n = 6), dogs (n = 3), hamsters (n = 3), and a ferret (n = 1) following experimental exposure. Five nonsynonymous changes relative to the USA-WA1/2020 prototype strain were near fixation in the stock used for inoculation but had reverted to wild-type sequences at these sites in dogs, cats, and hamsters within 1- to 3-d postexposure. A total of 14 emergent variants (six in nonstructural genes, six in spike, and one each in orf8 and nucleocapsid) were detected in viruses recovered from animals. This included substitutions in spike residues H69, N501, and D614, which also vary in human lineages of concern. Even though a live virus was not cultured from dogs, substitutions in replicase genes were detected in amplified sequences. The rapid selection of SARS-CoV-2 variants in vitro and in vivo reveals residues with functional significance during host switching. These observations also illustrate the potential for spillback from animal hosts to accelerate the evolution of new viral lineages, findings of particular concern for dogs and cats living in households with COVID-19 patients. More generally, this glimpse into viral host switching reveals the unrealized rapidity and plasticity of viral evolution in experimental animal model systems.

Cross-species transmission events, which challenge pathogens to survive in new host environments, typically result in species-specific adaptations (1). These evolutionary changes can determine the pathogenicity and transmissibility of the virus in novel host species (2). Pathogen host switching resulting in epidemic disease is a rare event that is constrained by the interaction between species (3). In contrast to most species, humans move globally and regularly come into contact with domestic and peridomestic animals. Thus, when a novel virus spreads through human populations, there is an incidental risk of exposure to potentially susceptible nonhuman species.This scenario has become evident with the SARS-CoV-2 pandemic (SI Appendix, Table S1). Originally resulting from viral spillover into humans (4, 5), likely from an animal reservoir, spillback into a wide range of companion and wild animals has occurred or been shown to be plausible (6–10), and an increasing number of studies have indicated a high frequency of human-to-animal SARS-CoV-2 spillback transmission (11–18). Given the short duration of viral shedding, serologic analyses present a more accurate characterization of actual animal exposures to SARS-CoV-2. Such studies conducted in a variety of animal species have illustrated surprisingly high levels of seroconversion in cats and dogs and more recently free-ranging deer (SI Appendix, Table S1) (73–87). Other well-documented spillback events include numerous mink farms (SI Appendix, Table S1). In one of these reports, multiple feral cats living on a mink farm in the Netherlands during a SARS-CoV-2 outbreak were seropositive, likely from the direct transmission of the virus from mink to cats, as owned cats on the same farm were seronegative (19). This further illustrates that cross-species transmission chains are readily achieved. Recent surveys of free-ranging white-tailed deer in Illinois, Michigan, New York, and Pennsylvania revealed 33% seropositivity in free-ranging animals (20). Active SARS-CoV-2 infection was subsequently confirmed by PCR in a deer in Ohio (21). Together, these findings suggest the likely establishment of multiple domestic animal and wildlife reservoirs of SARS-CoV-2.The repeated interspecies transmission of a virus presents the potential for the acceleration of viral evolution and a possible source of novel strain emergence. This was demonstrated by reverse zoonosis of SARS-CoV-2 from humans to mink, followed by a selection in mink and zoonotic transmission back to humans (8). Given that reverse zoonosis has been reported repeatedly in dogs and cats from households where COVID-19 patients reside, and the fact that up to 50% of households worldwide are inhabited by these companion animals, there is potential for similar transmission chains to arise via humans and their pets (22, 23). Elucidating the viral selection and species-specific adaptation of SARS-CoV-2 in common companion animals is therefore of high interest. Furthermore, understanding viral evolutionary patterns in both companion animals and experimental animal models provides a valuable appraisal of species-specific viral variants that spotlight genomic regions for host–virus interaction.Significant attention has been directed at substrains evolving from the initial SARS-CoV-2 isolate (24), and an accumulating number of variant lineages have demonstrated increased transmission potential in humans (25, 26). The role, if any, that reverse zoonotic infections of nonhuman species and spillback may have played in the emergence of these novel variants of SARS-CoV-2 remains unknown. Documenting viral evolution following the spillover of SARS-COV-2 into new species is difficult given the unpredictability of timing of these events; therefore, experimental studies can greatly aid the understanding of SARS-CoV-2 evolution in animal species. Laboratory-based studies also provide the opportunity to determine how changes that occur during viral expansion in cell culture may influence in vivo infections. This information is highly relevant for the interpretation of in vivo and in vitro experiments using inoculum propagated in culture.We therefore assessed the evolution of SARS-CoV-2 during the three rounds of expansion of strain USA-WA1/2020 in Vero E6 cells (27), followed by measuring the variant emergence occurring during primary experimental infection in four mammalian hosts. Specifically, we compared variant proportions, insertions, and deletions occurring in genomes of SARS-CoV-2 recovered from dogs (n = 3), cats (n = 6), hamsters (n = 3), and a ferret (n = 1).     

The neutralizing breadth of antibodies targeting diverse conserved epitopes between SARS-CoV and SARS-CoV-2

Antibody therapeutics for the treatment of COVID-19 have been highly successful. However, the recent emergence of the Omicron variant has posed a challenge, as it evades detection by most existing SARS-CoV-2 neutralizing antibodies (nAbs). Here, we successfully generated a panel of SARS-CoV-2/SARS-CoV cross-neutralizing antibodies by sequential immunization of the two pseudoviruses. Of the potential candidates, we found that nAbs X01, X10, and X17 offer broad neutralizing potential against most variants of concern, with X17 further identified as a Class 5 nAb with undiminished neutralization against the Omicron variant. Cryo-electron microscopy structures of the three antibodies together in complex with each of the spike proteins of the prototypical SARS-CoV, SARS-CoV-2, and Delta and Omicron variants of SARS-CoV-2 defined three nonoverlapping conserved epitopes on the receptor-binding domain. The triple-antibody mixture exhibited enhanced resistance to viral evasion and effective protection against infection of the Beta variant in hamsters. Our findings will aid the development of antibody therapeutics and broad vaccines against SARS-CoV-2 and its emerging variants.

By June 2022, the COVID-19 pandemic, caused by SARS-CoV-2, had resulted in more than 6 million deaths worldwide (1–3). Monoclonal antibodies (mAbs) isolated from SARS-CoV-2–infected individuals were effective as both therapeutic and prophylactic agents against SARS-CoV-2 (4–6), with several neutralizing antibodies (nAbs), including sotrovimab (7) and bamlanivimab (8), and nAb mixtures, including casirivimab–imdevimab (9) and bamlanivimab–etesevimab (10), approved under Emergency Use Authorization (EUA) for the treatment of patients with COVID-19. However, the constant evolution and genetic drift of SARS-CoV-2 has resulted in the emergence of many variants of concern (VOCs) depending on the main protein of the SARS-CoV-2 prototype strain, including the Alpha (B.1.1.7), Beta (B.1.351), Gamma (B.1.1.28), Delta (B.1.617.2), and Omicron (B.1.1.529) variants, the latter of which has become the major concern. Indeed, the Omicron variant harbors numerous residue substitutions in the spike (S) protein, with at least 15 mutations highly intertwined with common neutralizing epitopes in the receptor-binding domain (RBD) (11, 12). Various studies have reported that critical mutations within these VOCs prohibit the potent mAb neutralization that works against the ancestral isolate, leading to a much-diminished protective efficacy of antibody therapeutics against SARS-CoV-2 (13–20). Therefore, there is still a pressing need for nAbs with broader neutralizing breadth against current VOCs and future emerging variants.The trimeric S protein mediates SARS-CoV-2 entry into host cells via the RBD, which binds to the angiotensin-converting enzyme 2 (ACE2) receptor (1, 21, 22). Given its role, the RBD is regarded as a critical target for the development of therapeutics and vaccines against COVID-19. Indeed, numerous potently neutralizing mAbs are shown to target the receptor-binding motif (RBM) on the RBD, thereby efficiently inhibiting the S protein from binding to ACE2 to minimize or prohibit infection (4, 23, 24). However, VOCs frequently possess mutations within the RBM, which significantly reduces the neutralization breadth of mAbs that recognize this site (13–18, 25). Nevertheless, of the five classes of RBD-targeting nAbs (4, 26), three classes—represented by S309 (27), S2X259 (28), and S2H97 (26)—offer cross-neutralization against SARS-CoV-2 and SARS-CoV and thus can also inhibit infection from most VOCs. Consequently, it is assumed that epitopes within these sites are highly conserved among Sarbecoviruses and that antibody mixtures comprising representative nAbs that bind to these conserved epitopes may be able to prevent SARS-CoV-2 variants and other zoonotic “spillover” SARS-like viruses. In addition, under the selective pressure of antibody therapeutics, such as screening, the emergence of avoidance mutations becomes an important issue that should be considered. Such antibody avoidance studies in vitro have strongly supported the rationale of antibody mixtures consisting of noncompeting antibodies to avoid the development of resistance (13, 15, 29).nAbs reported to date have been primarily obtained from the human humoral immune response induced by vaccination or natural infection of SARS-CoV or SARS-CoV-2. The singular exposure of Sarbecoviruses at a time has hindered the generation of cross-neutralizing mAbs (26–28). Based on influenza virus research (30–32), the development of cross-neutralizing antibodies may benefit from the combined immunization of SARS-CoV and SARS-CoV-2 in sequence, offering insight into immune-focusing on conserved epitopes between the two virus strains.In this study, we focus on the conserved epitopes between SARS-CoV-2 and SARS-CoV. To this end, we generated a panel of broad-neutralizing antibodies (bnAbs) against SARS-CoV, SARS-CoV-2, and VOCs from sequentially immunized mice. Three representative bnAbs, X01, X10, and X17, were further identified to offer potent cross-neutralizing activity against most VOCs but with a decreased neutralization breadth against Omicron. High-resolution cryo-electron microscopy (cryo-EM) structures revealed three nonoverlapping conserved epitopes and defined the structural basis for the neutralization breadth of the three bnAbs. Using these three bnAbs in a mixture efficiently resisted viral escape and protected Syrian hamsters against challenge with the SARS-CoV-2 Beta variant. Thus, by taking advantage of conserved epitopes, our results expand upon the current therapeutic strategy and offer a way to cope with circulating and future emerging SARS-CoV-2 VOCs as well as any potential spillover zoonotic SARS-like viruses. This study thus highlights the potential utility of diverse, conserved epitopes for effective vaccine design.     

A highly efficacious live attenuated mumps virus–based SARS-CoV-2 vaccine candidate expressing a six-proline stabilized prefusion spike

With the rapid increase in SARS-CoV-2 cases in children, a safe and effective vaccine for this population is urgently needed. The MMR (measles/mumps/rubella) vaccine has been one of the safest and most effective human vaccines used in infants and children since the 1960s. Here, we developed live attenuated recombinant mumps virus (rMuV)–based SARS-CoV-2 vaccine candidates using the MuV Jeryl Lynn (JL2) vaccine strain backbone. The soluble prefusion SARS-CoV-2 spike protein (preS) gene, stablized by two prolines (preS-2P) or six prolines (preS-6P), was inserted into the MuV genome at the P–M or F–SH gene junctions in the MuV genome. preS-6P was more efficiently expressed than preS-2P, and preS-6P expression from the P–M gene junction was more efficient than from the F–SH gene junction. In mice, the rMuV-preS-6P vaccine was more immunogenic than the rMuV-preS-2P vaccine, eliciting stronger neutralizing antibodies and mucosal immunity. Sera raised in response to the rMuV-preS-6P vaccine neutralized SARS-CoV-2 variants of concern, including the Delta variant equivalently. Intranasal and/or subcutaneous immunization of IFNAR1^−/− mice and golden Syrian hamsters with the rMuV-preS-6P vaccine induced high levels of neutralizing antibodies, mucosal immunoglobulin A antibody, and T cell immune responses, and were completely protected from challenge by both SARS-CoV-2 USA-WA1/2020 and Delta variants. Therefore, rMuV-preS-6P is a highly promising COVID-19 vaccine candidate, warranting further development as a tetravalent MMR vaccine, which may include protection against SARS-CoV-2.

The current pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused tremendous damage to all aspects of our society (1–3). As of 1 June 2022, nearly 528 million cases have been reported worldwide, with nearly 6.3 million deaths (∼1.20% mortality). Symptoms of SARS-CoV-2 infection are primarily respiratory although increasing numbers of other syndromes such as cognitive deficits are being reported. As of June 2022, several SARS-CoV-2 vaccines based on messenger RNA (mRNA), inactivated virus, and adenovirus vectors (Ad26.COV2.S and ChAdOx1) have been approved for vaccination in humans over the age of 5 (4). These vaccines are highly efficacious, reaching 70 to 95% effectiveness against SARS-CoV-2 infection (4).Despite the high success of the current SARS-CoV-2 vaccines, there are several limitations. Protection provided by current vaccines begins to decline after 3 mo (5), which has required a third or fourth dose to boost the immune response. Current vaccines are less effective against recently emergent SARS-CoV-2 variants of concern (VoCs) (6–9). More and more evidence has shown that vaccine-induced neutralizing antibodies were significantly weakened or insufficient to neutralize VoCs such as the Delta variant (7–9), which spreads much faster and causes more severe illness than the earlier strains. In addition, the current vaccines neutralize the most recently emerged variant, Omicron, ∼40 times less efficiently compared with early SARS-CoV-2 isolates (10, 11). The mRNA vaccines are expensive to produce, hard to transport internationally, and difficult to store in many countries because of the requirement for expensive −80 °C freezers.A safe and efficacious pediatric SARS-CoV-2 vaccine is needed to halt the current pandemic. Pfizer’s mRNA vaccine is 90.7% effective in preventing COVID-19 symptoms in children 5 to 11 y old (12, 13). On 17 June 2022, Food and Drug Administration (FDA) authorized emergency use of the Moderna and Pfizer mRNA vaccines for children down to 6 mo of age. As of 23 June 2022, a total of 13.7 million COVID-19 cases have occurred in children, representing 18.8% of the total COVID-19 cases in the United States. Notably, COVID-19 cases in children have increased significantly after the reopening of schools. Therefore, development of other vaccine platforms and strategies to enhance durability, reduce cost, and enhance stability are essential for terminating the pandemic.Historically, the MMR (measles/mumps/rubella) vaccine has been one of the safest and most effective human vaccines ever developed (14–16). The application in children started in the 1960s and provides long-lasting protection against these three viruses (14, 16). Among the three MMR components, measles virus (MeV) and mumps virus (MuV) are nonsegmented negative-sense (NNS) RNA viruses, belonging to the family Paramyxoviridae in the order Mononegavirales. The MuV genome is 15,384 nt in length, and it encodes seven structural proteins arranged in the order 3′-leader-N-P-M-F-SH-HN-L-trailer-5′ (17). The limited number of discrete genes of the NNS RNA genome and the intergenic regions available for inserting additional genes facilitates the development of live vectored vaccines. MuV is an excellent viral vector for delivery of vaccines against other highly pathogenic viruses, primarily because of its high safety and efficacy, well-established good manufacturing practices, induction of long-lived immunity, and the potential for the development of a quadrivalent vaccine against four major pediatric diseases (18, 19).In this study, we developed a suite of safe and highly efficacious recombinant MuV (rMuV)–based SARS-CoV-2 vaccine candidates expressing a stabilized prefusion spike with two prolines (preS-2P) or six prolines (preS-6P) at different gene junctions in the MuV genome. Among them, the rMuV-based preS-6P vaccine induces a broad neutralizing antibody against VoCs and T cell immunity, and provides complete protection against SARS-CoV-2 WA1 and the Delta variant challenge in animal models.     

SARS-CoV-2 spreads through cell-to-cell transmission

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible coronavirus responsible for the global COVID-19 pandemic. Herein, we provide evidence that SARS-CoV-2 spreads through cell–cell contact in cultures, mediated by the spike glycoprotein. SARS-CoV-2 spike is more efficient in facilitating cell-to-cell transmission than is SARS-CoV spike, which reflects, in part, their differential cell–cell fusion activity. Interestingly, treatment of cocultured cells with endosomal entry inhibitors impairs cell-to-cell transmission, implicating endosomal membrane fusion as an underlying mechanism. Compared with cell-free infection, cell-to-cell transmission of SARS-CoV-2 is refractory to inhibition by neutralizing antibody or convalescent sera of COVID-19 patients. While angiotensin-converting enzyme 2 enhances cell-to-cell transmission, we find that it is not absolutely required. Notably, despite differences in cell-free infectivity, the authentic variants of concern (VOCs) B.1.1.7 (alpha) and B.1.351 (beta) have similar cell-to-cell transmission capability. Moreover, B.1.351 is more resistant to neutralization by vaccinee sera in cell-free infection, whereas B.1.1.7 is more resistant to inhibition by vaccinee sera in cell-to-cell transmission. Overall, our study reveals critical features of SARS-CoV-2 spike-mediated cell-to-cell transmission, with important implications for a better understanding of SARS-CoV-2 spread and pathogenesis.

SARS-CoV-2 is a novel beta-coronavirus that is closely related to two other highly pathogenic human coronaviruses, SARS-CoV and MERS-CoV (1). The spike (S) proteins of SARS-CoV-2 and SARS-CoV mediate entry into target cells, and both use angiotensin-converting enzyme 2 (ACE2) as the primary receptor (2–6). The spike protein of SARS-CoV-2 is also responsible for induction of neutralizing antibodies, thus playing a critical role in host immunity to viral infection (7–10).Similar to HIV and other class I viral fusion proteins, SARS-CoV-2 spike is synthesized as a precursor that is subsequently cleaved and highly glycosylated; these properties are critical for regulating viral fusion activation, native spike structure, and evasion of host immunity (11–15). However, distinct from SARS-CoV, yet similar to MERS-CoV, the spike protein of SARS-CoV-2 is cleaved by furin into S1 and S2 subunits during the maturation process in producer cells (6, 16, 17). S1 is responsible for binding to the ACE2 receptor, whereas S2 mediates viral membrane fusion (18, 19). SARS-CoV-2 spike can also be cleaved by additional host proteases, including transmembrane serine protease 2 (TMPRSS2) on the plasma membrane and several cathepsins in the endosome, which facilitate viral membrane fusion and entry into host cells (20–22).Enveloped viruses spread in cultured cells and tissues via two routes: by cell-free particles and through cell–cell contact (23–26). The latter mode of viral transmission normally involves tight cell–cell contacts, sometimes forming virological synapses, where local viral particle density increases (27), resulting in efficient transfer of virus to neighboring cells (24). Additionally, cell-to-cell transmission has the ability to evade antibody neutralization, accounting for efficient virus spread and pathogenesis, as has been shown for HIV and hepatitis C virus (HCV) (28–32). Low levels of neutralizing antibodies, as well as a deficiency in type I IFNs, have been reported for SARS-CoV-2 (18, 33–37) and may have contributed to the COVID-19 pandemic and disease progression (38–43).In this work, we evaluated cell-to-cell transmission of SARS-CoV-2 in the context of cell-free infection and in comparison with SARS-CoV. Results from this in vitro study reveal the heretofore unrecognized role of cell-to-cell transmission that potentially impacts SARS-CoV-2 spread, pathogenesis, and shielding from antibodies in vivo.     

Engineered SARS-CoV-2 receptor binding domain improves manufacturability in yeast and immunogenicity in mice

Global containment of COVID-19 still requires accessible and affordable vaccines for low- and middle-income countries (LMICs). Recently approved vaccines provide needed interventions, albeit at prices that may limit their global access. Subunit vaccines based on recombinant proteins are suited for large-volume microbial manufacturing to yield billions of doses annually, minimizing their manufacturing cost. These types of vaccines are well-established, proven interventions with multiple safe and efficacious commercial examples. Many vaccine candidates of this type for SARS-CoV-2 rely on sequences containing the receptor-binding domain (RBD), which mediates viral entry to cells via ACE2. Here we report an engineered sequence variant of RBD that exhibits high-yield manufacturability, high-affinity binding to ACE2, and enhanced immunogenicity after a single dose in mice compared to the Wuhan-Hu-1 variant used in current vaccines. Antibodies raised against the engineered protein exhibited heterotypic binding to the RBD from two recently reported SARS-CoV-2 variants of concern (501Y.V1/V2). Presentation of the engineered RBD on a designed virus-like particle (VLP) also reduced weight loss in hamsters upon viral challenge.

Prevention of COVID-19 on a global scale will require >10 billion doses of vaccines for SARS-CoV-2; most of which are needed in low- and middle-income countries (LMICs) (1). To ensure adequate supply and global access, vaccine manufacturers must select highly immunogenic vaccine antigens that offer broad protection against emerging variants and are compatible with large-volume production in existing manufacturing facilities (2, 3). Vaccines using mRNA have established the efficacy of vaccines for SARS-CoV-2 based on full-length trimeric spike (S) protein (4, 5). Recombinant S protein produced in mammalian or insect cells has also shown immunogenicity and efficacy in nonhuman primates (6). Protein-based vaccines hold promise for large-volume, low-cost production, and are safe and efficacious (7–9). The receptor-binding domain (RBD) subunit, which mediates viral entry to cells via ACE2 (10–12), has emerged as an important alternative antigen (13). Antibodies to RBD account for most of the neutralizing activity elicited in natural infections, and several potent monoclonal antibodies have been discovered from convalescent patients (14, 15). A His-tagged SARS-CoV-2 RBD construct based on SARS-CoV-2 Wuhan-Hu-1 and produced in insect cells has elicited neutralizing antibodies in mice and protective immunity in nonhuman primates (16). Similar tagged constructs have also been adapted for production in yeast like Komagataella phaffii (Pichia pastoris) (17, 18), establishing the RBD domain as a prominent candidate for large-volume manufacturing of COVID-19 vaccines.Despite its significance for low-cost vaccine candidates, recombinant RBD based on the original SARS-CoV-2 clade 19A sequence has shown limited immunogenicity to date. Reported candidates would require as many as three doses or large doses to elicit strong neutralizing antibody responses in mice when formulated with adjuvants (16, 18). Increasing the number of doses or amounts required could limit its benefits for affordable and accessible vaccines. An engineered design for the RBD, therefore, could enhance the potency of many subunit-based vaccine candidates using this domain.     

A novel class of TMPRSS2 inhibitors potently block SARS-CoV-2 and MERS-CoV viral entry and protect human epithelial lung cells

The host cell serine protease TMPRSS2 is an attractive therapeutic target for COVID-19 drug discovery. This protease activates the Spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and of other coronaviruses and is essential for viral spread in the lung. Utilizing rational structure-based drug design (SBDD) coupled to substrate specificity screening of TMPRSS2, we have discovered covalent small-molecule ketobenzothiazole (kbt) TMPRSS2 inhibitors which are structurally distinct from and have significantly improved activity over the existing known inhibitors Camostat and Nafamostat. Lead compound MM3122 (4) has an IC₅₀ (half-maximal inhibitory concentration) of 340 pM against recombinant full-length TMPRSS2 protein, an EC₅₀ (half-maximal effective concentration) of 430 pM in blocking host cell entry into Calu-3 human lung epithelial cells of a newly developed VSV-SARS-CoV-2 chimeric virus, and an EC₅₀ of 74 nM in inhibiting cytopathic effects induced by SARS-CoV-2 virus in Calu-3 cells. Further, MM3122 blocks Middle East respiratory syndrome coronavirus (MERS-CoV) cell entry with an EC₅₀ of 870 pM. MM3122 has excellent metabolic stability, safety, and pharmacokinetics in mice, with a half-life of 8.6 h in plasma and 7.5 h in lung tissue, making it suitable for in vivo efficacy evaluation and a promising drug candidate for COVID-19 treatment.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the newly emerged, highly transmissible coronavirus responsible for the ongoing COVID-19 pandemic, which is associated with 136 million cases and almost 3 million deaths worldwide as of April 12, 2021 (https://coronavirus.jhu.edu/map.html). While three vaccines have recently been approved by the FDA, there are still no clinically approved small-molecule drugs available for the treatment of this disease except Remdesivir, and the effectiveness of the vaccines against immune escape variants might be reduced. Multiple therapeutic strategies have been proposed (1−2), including both viral and host proteins, but none have yet been fully validated for clinical application. One class of protein targets which have shown promising results are proteolytic enzymes including the viral proteases (1, 3–5), Papain-Like Protease (PLpro) and the 3C-like or “Main Protease” (3CL or MPro), and several host proteases involved in viral entry, replication, and effects on the immune system creating the life-threatening symptoms of COVID-19 infection (4–6). The latter include various members of the cathepsin family of cysteine proteases, including cathepsin L, furin, and the serine proteases factor Xa, plasmin, elastase, tryptase, TMPRSS2, and TMPRSS4.Coronavirus (SARS CoV-2, SARS-CoV, and Middle East respiratory syndrome coronavirus [MERS]) entry is mediated by the viral spike protein, which must be cleaved by host proteases in order to trigger membrane fusion and entry into the host cell after binding to the host cell receptor Angiotensin Converting Enzyme-2 (ACE2) (7–10). This is mediated by initial cleavage at the S1/S2 junction of spike, which is thought to occur during processing in the producer cell, followed by cleavage at the S2′ site either by serine proteases at the cell surface or by cathepsin proteases in the late endosome or endolysosome (9, 10). Whether serine or cathepsin proteases are used for S2′ cleavage is cell type dependent. While entry into Calu-3 (human lung epithelial) or HAE (primarily human airway epithelial) cells is cathepsin independent, entry into Vero cells (African green monkey kidney epithelial), which do not express the required serine proteases, depends exclusively on cathepsins (7, 9–11).TMPRSS2 (12) is a type II transmembrane serine proteases (TTSP) (13) that has been shown to be crucial for host cell viral entry and spread of SARS-CoV-2 (7, 8, 14–16), as well as SARS-CoV (17, 18), MERS-CoV (19), and influenza A viruses (20–27). The Spike protein requires proteolytic processing/priming by TMPRSS2 to mediate entry into lung cells; thus, small-molecule inhibitors of this target offer much promise as new therapeutics for COVID-19 and other coronavirus diseases (7, 8). TMPRSS2 expression levels dictate the entry route used by SARS-CoV-2 to enter cells, as reported recently (28). In cells that express little or no TMPRSS2, cell entry occurs via the endosomal pathway, and cleavage of spike protein is performed by cathepsin L. It has been demonstrated that the TMPRSS2-expressing lung epithelial Calu-3 cells are highly permissive to SARS-CoV-2 infection. The irreversible serine protease inhibitors Camostat (7) and Nafamostat (29) are effective at preventing host cell entry and replication of SARS-CoV-2 in Calu-3 cells through a TMPRSS2-dependent mechanism (14, 15).Herein, we report on the discovery of a class of substrate-based ketobenzothiazole (kbt) inhibitors of TMPRSS2 with potent antiviral activity against SARS-CoV-2 which are significantly improved over Camostat and Nafamostat. Several compounds were found to be strong inhibitors of viral entry and replication, with EC₅₀ (half-maximal effective concentration) values exceeding the potency of Camostat and Nafamostat and without cytotoxicity. Newly developed compound MM3122 (4) has excellent pharmacokinetics (PK) and safety in mice and is thus a promising lead candidate drug for COVID-19 treatment.     

Nonmuscle myosin heavy chain IIA facilitates SARS-CoV-2 infection in human pulmonary cells

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), binds to host receptor angiotensin-converting enzyme 2 (ACE2) through its spike (S) glycoprotein, which mediates membrane fusion and viral entry. However, the expression of ACE2 is extremely low in a variety of human tissues, especially in the airways. Thus, other coreceptors and/or cofactors on the surface of host cells may contribute to SARS-CoV-2 infection. Here, we identified nonmuscle myosin heavy chain IIA (MYH9) as an important host factor for SARS-CoV-2 infection of human pulmonary cells by using APEX2 proximity-labeling techniques. Genetic ablation of MYH9 significantly reduced SARS-CoV-2 pseudovirus infection in wild type (WT) A549 and Calu-3 cells, and overexpression of MYH9 enhanced the pseudovirus infection in WT A549 and H1299 cells. MYH9 was colocalized with the SARS-CoV-2 S and directly interacted with SARS-CoV-2 S through the S2 subunit and S1-NTD (N-terminal domain) by its C-terminal domain (designated as PRA). Further experiments suggested that endosomal or myosin inhibitors effectively block the viral entry of SARS-CoV-2 into PRA-A549 cells, while transmembrane protease serine 2 (TMPRSS2) and cathepsin B and L (CatB/L) inhibitors do not, indicating that MYH9 promotes SARS-CoV-2 endocytosis and bypasses TMPRSS2 and CatB/L pathway. Finally, we demonstrated that loss of MYH9 reduces authentic SARS-CoV-2 infection in Calu-3, ACE2-A549, and ACE2-H1299 cells. Together, our results suggest that MYH9 is a candidate host factor for SARS-CoV-2, which mediates the virus entering host cells by endocytosis in an ACE2-dependent manner, and may serve as a potential target for future clinical intervention strategies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered a pandemic coronavirus disease (COVID-19) (1–3), resulting in more than 209 million infections worldwide since December 2019. SARS-CoV-2 is the seventh coronavirus (CoV) infecting humans beyond endemic human CoVs (HCoV-HKU1, -NL63, -OC43, and -229E), SARS-CoV, and Middle East respiratory syndrome CoV (MERS-CoV). SARS-CoV-2 preferentially infects the airway epithelial cells (1, 4), although viral infection has been detected in a variety of human organs, including the lungs, pharynx, heart, liver, brain, kidneys, and digestive system organs (5–7), causing upper respiratory diseases, fever, and severe pneumonia in humans.CoVs were not considered to be highly pathogenic to humans until the outbreak of SARS-CoV started in 2002 in Guangdong province, China (8–10). The primary determinant of CoV tropism is their spike (S) glycoproteins, which mediate the viral infection by binding to membrane receptors on the host cells and could be cleaved to the N-terminal S1 and C-terminal S2 subunit by the host proteases such as transmembrane protease serine 2 (TMPRSS2) and Furin (11). Studies have shown that ACE2, a cellular receptor for SARS-CoV, also binds SARS-CoV-2 S and serves as the entry point for SARS-CoV-2 (3, 12). However, ACE2 cannot fully explain the tissue tropism of SARS-CoV-2 because of the virus detection in tissues with little ACE2 expression, such as the liver, brain, and blood, and even in the lung, only a small subset of cells expresses ACE2 (SI Appendix, Fig. 1B) (13–16). Recently, neuropilin-1 (NRP1) (17, 18) and heparan sulfate (19, 20) have been identified as cofactors implicated in enhancing ACE2-dependent SARS-CoV-2 infection, while tyrosine-protein kinase receptor (AXL) (21) and CD147 (22) were identified as receptors involved in SARS-CoV-2 cell entry independently of ACE2. These highlight the multiple routes of SARS-CoV-2 infection, likely contributing to high infectivity and spread of COVID-19. Hence, SARS-CoV-2 is assumed to be dependent on other potential receptors or coreceptors to facilitate its infection in humans.Here, we applied engineered ascorbate peroxidase APEX2-based subcellular proteomics (23–25) to capture spatiotemporal membrane protein complexes interacting with SARS-CoV-2 S in living cells. We found that the nonmuscle myosin heavy chain IIA (MYH9) specifically interacts with SARS-CoV-2 S protein. The C-terminal domain (designated as PRA) of MYH9 interacts with the S2 subunit and the N-terminal domain (NTD) of the S1 subunit, and PRA overexpression facilitates a pan-CoV entry into host cells. In addition, depletion of MYH9 significantly reduced authentic SARS-CoV-2 infection in human lung cell lines. Mechanistically, a myosin inhibitor but not TMPRSS2 and CatB/L inhibitors blocked SARS-CoV-2 infection in PRA-A549 cells, and the presence of ACE2 is required for MYH9 mediated SARS-CoV-2 entry. Collectively, our results suggest that MYH9 is a coreceptor of ACE2 for SARS-CoV-2 infection in human pulmonary cells.     

Inhalable nanocatchers for SARS-CoV-2 inhibition

The global coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome (SARS)–like coronavirus (SARS-CoV-2), presents an urgent health crisis. More recently, an increasing number of mutated strains of SARS-CoV-2 have been identified globally. Such mutations, especially those on the spike glycoprotein to render its higher binding affinity to human angiotensin-converting enzyme II (hACE2) receptors, not only resulted in higher transmission of SARS-CoV-2 but also raised serious concerns regarding the efficacies of vaccines against mutated viruses. Since ACE2 is the virus-binding protein on human cells regardless of viral mutations, we design hACE2-containing nanocatchers (NCs) as the competitor with host cells for virus binding to protect cells from SARS-CoV-2 infection. The hACE2-containing NCs, derived from the cellular membrane of genetically engineered cells stably expressing hACE2, exhibited excellent neutralization ability against pseudoviruses of both wild-type SARS-CoV-2 and the D614G variant. To prevent SARS-CoV-2 infections in the lung, the most vulnerable organ for COVID-19, we develop an inhalable formulation by mixing hACE2-containing NCs with mucoadhesive excipient hyaluronic acid, the latter of which could significantly prolong the retention of NCs in the lung after inhalation. Excitingly, inhalation of our formulation could lead to potent pseudovirus inhibition ability in hACE2-expressing mouse model, without imposing any appreciable side effects. Importantly, our inhalable hACE2-containing NCs in the lyophilized formulation would allow long-term storage, facilitating their future clinical use. Thus, this work may provide an alternative tactic to inhibit SARS-CoV-2 infections even with different mutations, exhibiting great potential for treatment of the ongoing COVID-19 epidemic.

The expanding coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected more than 200 million people and killed over 3 million, and the numbers are still rapidly rising in April 2021 (1, 2). Like other zoonotic coronaviruses, SARS-CoV-2 with the surface spike (S) glycoprotein binds with the receptor human angiotensin-converting enzyme II (hACE2) for cell entry and infection (3–6). Moreover, the S protein also undergoes mutations all the time to optimize its binding affinity and binding mode with ACE2 receptors, which may alter pathogenesis, virulence, and transmissibility (7–9). The spike aspartic acid–614 to glycine (D614G), the dominant SARS-CoV-2 mutational form globally, showed obviously increased binding efficiency with ACE2 receptor during the virus infection process (10, 11). Moreover, a new variant strain of SARS-CoV-2, B.1.1.7 (also known as VOC 202012/01), with a high number of genetic mutations has been found in London and is spreading worldwide (12, 13). The binding affinity of S protein of B.1.1.7 to hACE2 receptor is increased by 1,000 times, and it exhibits 70% more transmissible ability than the previously discovered SARS-CoV-2 (14).Vaccines, one of the effective strategies to prevent the spread of infectious diseases by reducing morbidity and mortality, have attracted wide attention since the outbreak of COVID-19. At present, there are more than 314 SARS-CoV-2 vaccines in the research and development stage in the world, 89 of which have entered the clinical trial stage, and multiple research and development technologies are being promoted in parallel to promote vaccine development (15). Excitingly, thirteen vaccines have been approved for clinical use in different countries, including mRNA vaccines, virus-vectored vaccines, inactivated virus vaccines, and protein subunit vaccines (https://vac-lshtm.shinyapps.io/ncov_vaccine_landscape/#https://vac-lshtm.shinyapps.io/ncov_vaccine_landscape/). The current vaccines primarily protect the host against infection by producing neutralizing antibodies specific for the surface S protein (16–18). However, mutation of the S protein may possibly limit the efficiency of these vaccines (19–21). For example, the neutralizing activity of the serum of volunteers who have received either the Moderna (mRNA-1273) or Pfizer-BioNTech (BNT162b2) vaccines against the South Africa mutant strain (B.1.351) have been proven to be reduced (22, 23). Thus, novel strategies that could effectively and rapidly prevent the infection of SARS-CoV-2 with different mutations take on a renewed urgency in this period for COVID-19.Considering that the infectivity of SARS-CoV-2 depends on the binding with the entry receptor hACE2, recent studies have investigated the potential of cellular nanovesicles (NVs) containing hACE2 to compete with host cells for SARS-CoV-2 binding to protect host cells from the infection of SARS-CoV-2 (24, 25). How to improve the therapeutic efficiency of such neutralizing cellular NVs is still an important issue for COVID-19 treatment. Herein, the hACE2 nanocatchers (NCs) were fabricated from genetically engineering human embryonic kidney 293T cells with hACE2 as the neutralizing NCs to protect the host from the infection of SARS-CoV-2. As expected, such hACE2-containing NCs displayed excellent binding affinity to the coronavirus and its mutant and inhibited their usual infection ability in vitro. Moreover, in view of the fact that the effective retention of cellular NVs in the lungs after inhalation may be a prerequisite for inhibiting SARS-CoV-2 infection of the lung, we try to seek effective mucoadhesives to increase epithelial contact, decrease mucociliary transport rate, and finally, maintain the retention of NCs in the lungs for a longer duration after pulmonary drug delivery. Excitingly, it was found that the mucoadhesive excipient hyaluronic acid (HA) with high biocompatibility introduced here was able to significantly improve the retention of NCs in the lung, exhibiting potent SARS-CoV-2 pseudovirus inhibition ability in the mouse model with replication defective adenovirus encoding for hACE2. More importantly, NCs in the lyophilized formulation were fabricated with the assistance of cryoprotectant sucrose, increasing the feasibility of clinical use including transport and long-term storage.