Immunogenicity of rVSVΔG-ZEBOV-GP Ebola vaccination in exposed and potentially exposed persons in the Democratic Republic of the Congo

Despite more than 300,000 rVSVΔG-ZEBOV-glycoprotein (GP) vaccine doses having been administered during Ebola virus disease (EVD) outbreaks in the Democratic Republic of the Congo (DRC) between 2018 and 2020, seroepidemiologic studies of vaccinated Congolese populations are lacking. This study examines the antibody response at 21 d and 6 mo postvaccination after single-dose rVSVΔG-ZEBOV-GP vaccination among EVD-exposed and potentially exposed populations in the DRC. We conducted a longitudinal cohort study of 608 rVSVΔG-ZEBOV-GP–vaccinated individuals during an EVD outbreak in North Kivu Province, DRC. Participants provided questionnaires and blood samples at three study visits (day 0, visit 1; day 21, visit 2; and month 6, visit 3). Anti-GP immunoglobulin G (IgG) antibody titers were measured in serum by the Filovirus Animal Nonclinical Group anti-Ebola virus GP IgG enzyme-linked immunosorbent assay. Antibody response was defined as an antibody titer that had increased fourfold from visit 1 to visit 2 and was above four times the lower limit of quantification at visit 2; antibody persistence was defined as a similar increase from visit 1 to visit 3. We then examined demographics for associations with follow-up antibody titers using generalized linear mixed models. A majority of the sample, 87.2%, had an antibody response at visit 2, and 95.6% demonstrated antibody persistence at visit 3. Being female and of young age was predictive of a higher antibody titer postvaccination. Antibody response and persistence after Ebola vaccination was robust in this cohort, confirming findings from outside of the DRC.

Since the rVSVΔG-ZEBOV-glycoprotein (GP) vaccine completed clinical trials in West Africa, over 300,000 doses of the vaccine have been deployed in response to the multiple Ebola virus disease (EVD) outbreaks in the Democratic Republic of the Congo (DRC). While initially deployed under a “compassionate use/expanded access” protocol (1, 2), as of December 19, 2019, the vaccine was officially licensed by both the American (Food and Drug Administration, FDA) and European (European Medicines Agency) regulatory agencies (3, 4). Wide use of this vaccine was supported by evidence gathered in clinical trials and other studies, including those postlicensure conducted in North America and West Africa, which demonstrated short-term vaccine efficacy (5–16). In addition to short-term protection, clinical trials and other studies have provided evidence of Ebolavirus Zaire (EBOV)–specific antibody persistence up to 2 y postvaccination, suggesting that the vaccine may continue to offer protective immunity over time (5, 7, 8, 14, 15). While promising, observations of successful rVSVΔG-ZEBOV-GP vaccine performance in outbreak settings have mostly come from studies conducted at the end of the 2014 to 2016 West African EVD outbreak (7, 13, 14, 17). Such studies in the DRC are lacking.Furthermore, recent evidence of breakthrough infections within the DRC has highlighted the need for DRC-specific vaccine research, including magnitude and durability of serological response after rVSVΔG-ZEBOV-GP vaccination in Congolese populations. In April 2019, the World Health Organization (WHO) released a preliminary report of rVSVΔG-ZEBOV-GP efficacy in the 2018 to 2020 Beni outbreak. Among 93,965 people at risk who were vaccinated, there were 15 confirmed EVD cases with onset of symptoms 10 d or more postvaccination (18). Another report describes an individual who presented with EVD 6 mo after vaccination, initiating a chain of transmission resulting in 91 subsequent infections (19), prompting questions around the duration of protection. These recent events highlight both the consequences of breakthrough infections and the possibility of waning immunity postvaccination.When considering rVSVΔG-ZEBOV-GP performance in the DRC, there are several factors that may impact the effect of vaccination in Congolese populations. First, an increase in vaccination dose could have resulted in increased immunogenicity in the DRC. Vaccination deployment during the EVD outbreak of 2018 initially included double the plaque-forming units (PFUs) in the vaccine dosage compared to what was used in West Africa (20 million PFU/mL versus 10 million PFU/mL, respectively) (20). As previous studies have identified varying immunogenicity after different vaccine doses in different locations, this variation in vaccine dose could lead to differing antibody responses from previously studied cohorts (15). Second, an important component of the vaccine deployment was the requirement for an ultracold chain (storage of vaccine at −70 °C), which poses extreme logistical challenges in resource-constrained environments. Despite considerable efforts to avoid cold chain failures, it is plausible that fluctuations could have occurred and caused changes in vaccine effectiveness (21). Third, populations in this region may have a baseline level of filovirus seroreactivity that may enable a more robust response to Ebola vaccination (15, 22). Previous serologic studies in the DRC have indicated that Congolese populations may not be naive to filovirus exposures, with individuals presenting evidence of robust antibody responses to various filoviruses in the absence of a known history of EVD (23–26). While there had never been a reported EVD outbreak in North Kivu prior to 2018, this province is known for highly mobile populations; proximity to large forested areas, which may harbor filovirus or filovirus-like pathogens; and access to cross-border populations, including those from Uganda, which have had previous filovirus outbreaks (27–29). Finally, the underlying prevalence of immunosuppressive conditions, such as HIV infection and poor nutritional status, could hinder vaccine immunogenicity in Congolese populations (30).Given the DRC’s unique landscape, which includes evidence of breakthrough infections, a more thorough region-specific understanding of serologic response to Ebola vaccination is needed. Various factors such as vaccine dose, storage conditions, current infections, and previous exposure may alter the magnitude and durability of antibody response after vaccination in Congolese populations (7, 31, 32). To better understand rVSVΔG-ZEBOV-GP performance in the DRC, we conducted a seroepidemiologic study of postvaccination antibody persistence in Congolese populations, who may have meaningfully different experiences than those in West Africa. Here, we provide a preliminary report of antibody response and persistence, along with potential predictors, after single-dose rVSVΔG-ZEBOV-GP vaccination among EVD-exposed and potentially exposed populations in the DRC.     

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.     

From the Cover: Preexisting CD8+ T-cell immunity to the H7N9 influenza A virus varies across ethnicities

The absence of preexisting neutralizing antibodies specific for the novel A (H7N9) influenza virus indicates a lack of prior human exposure. As influenza A virus–specific CD8⁺ T lymphocytes (CTLs) can be broadly cross-reactive, we tested whether immunogenic peptides derived from H7N9 might be recognized by memory CTLs established following infection with other influenza strains. Probing across multiple ethnicities, we identified 32 conserved epitopes derived from the nucleoprotein (NP) and matrix-1 (M1) proteins. These NP and M1 peptides are presented by HLAs prevalent in 16–57% of individuals. Remarkably, some HLA alleles (A*0201, A*0301, B*5701_, B*1801, and B*0801) elicit robust CTL responses against any human influenza A virus, including H7N9, whereas ethnicities where HLA-A*0101, A*6801, B*1501, and A*2402 are prominent, show limited CTL response profiles. By this criterion, some groups, especially the Alaskan and Australian Indigenous peoples, would be particularly vulnerable to H7N9 infection. This dissection of CTL-mediated immunity to H7N9 thus suggests strategies for both vaccine delivery and development.Emerging unexpectedly in February 2013, the H7N9 influenza A virus (IAV) has thus far caused 137 human infections with 45 deaths (1). Clinical manifestations include major respiratory compromise, multiorgan failure, and exceedingly high serum cytokine and chemokine levels (2). Although May through September saw only five such cases, two more were recorded in October (1), indicating that H7N9 may return during the northern winter. Furthermore, the presence of a natural avian reservoir and the severity of the disease emphasized the need to focus on protective immunity. Most patients had contact with poultry within a week before clinical onset (2), suggesting that domestic birds are the source (2, 3). Even so, the potential for person-to-person spread is highlighted by ferret experiments (4) and instances of infection via close family contact (3). A very real concern is that further mutations may facilitate human-to-human transmission (5).Evidence from animal (6) and human studies (7–9) suggests that, in the absence of neutralizing antibodies (NAbs), preexisting memory CD8⁺ T lymphocytes (CTLs) directed at conserved and/or cross-reactive IAV peptide + class I HLA (pHLA1) epitopes can diminish disease severity. The recall of IAV-specific CTLs promotes recovery manifested by milder symptoms, diminished virus shedding and transmission (6, 7). A comprehensive analysis of the 2009 pandemic H1N1 IAV (H1N1pdm-2009) indicated that CTL memory provided some protection for the antibody naïve (9). Thus, cross-reactive CTL memory generated after a prior encounter with seasonal or pandemic IAVs, or by a CTL-directed vaccine, could potentially limit the severity of an H7N9 pandemic.The present analysis probes the extent of preexisting CTL immunity in populations that have not been exposed to the H7N9 virus. This potential for CTL recall is defined for HLA1s that are differentially prominent in various ethnicities. Using an evolutionary and immunological approach, we show substantial levels of immunogenic peptide conservation for nucleoprotein (NP) and matrix-1 (M1), with estimated coverage according to known HLA1 presentation profiles ranging between 16% and 57% of the global population. Overall, the findings support the view that it is important to consider developing vaccines with a T cell–based component that has the potential to protect against severe novel IAV infections. Furthermore, given that some ethnicities, including Australia’s Indigenous and Alaskan people, show evidence of a diminished HLA1-related response capacity, it is essential that health policy development and planning gives such groups priority in IAV vaccination campaigns. The 2009 H1N1 pandemic caused higher attack rates and morbidity among Indigenous populations in the Americas, New Zealand, and Australia (10).     

Induction of broadly cross-reactive antibody responses to the influenza HA stem region following H5N1 vaccination in humans

The emergence of pandemic influenza viruses poses a major public health threat. Therefore, there is a need for a vaccine that can induce broadly cross-reactive antibodies that protect against seasonal as well as pandemic influenza strains. Human broadly neutralizing antibodies directed against highly conserved epitopes in the stem region of influenza virus HA have been recently characterized. However, it remains unknown what the baseline levels are of antibodies and memory B cells that are directed against these conserved epitopes. More importantly, it is also not known to what extent anti-HA stem B-cell responses get boosted in humans after seasonal influenza vaccination. In this study, we have addressed these two outstanding questions. Our data show that: (i) antibodies and memory B cells directed against the conserved HA stem region are prevalent in humans, but their levels are much lower than B-cell responses directed to variable epitopes in the HA head; (ii) current seasonal influenza vaccines are efficient in inducing B-cell responses to the variable HA head region but they fail to boost responses to the conserved HA stem region; and (iii) in striking contrast, immunization of humans with the avian influenza virus H5N1 induced broadly cross-reactive HA stem-specific antibodies. Taken together, our findings provide a potential vaccination strategy where heterologous influenza immunization could be used for increasing the levels of broadly neutralizing antibodies and for priming the human population to respond quickly to emerging pandemic influenza threats.The emergence of novel influenza virus strains poses a continuous public health threat (1, 2). The World Health Organization estimates that influenza viruses infect one-billion people annually, with three- to five-million cases of severe illness, and up to 500,000 deaths worldwide (3). Following influenza virus infection, humoral immune responses against the viral hemagglutinin (HA) protein may persist for decades in humans (4). These anti-HA responses correlate strongly with protection against influenza infection (5). Serological memory is maintained by antibody-secreting long-lived plasma cells and reinforced by memory B cells, which can rapidly differentiate into antibody-secreting cells upon antigen reexposure (6).Influenza vaccine efficacy is constantly undermined by antigenic variation in the circulating viral strains, particularly in the HA and neuraminidase (NA) proteins. Current influenza vaccination strategies rely on changing the HA and NA components of the annual human vaccine to ensure that they antigenically match circulating influenza strains (7, 8). Developing an influenza vaccine that is capable of providing broad and long-lasting protective antibody responses remains the central challenge for influenza virus research.HA is a trimer, with each monomer comprised of two subunits: HA1, which includes the HA globular head, and HA2, whose ectodomain together with the N- and C-terminal parts of HA1 constitute the HA stem region (9). Phylogenetically, the 18 HA subtypes characterized so far are divided into two groups. Among strains that have recently caused disease in humans, H1 and H5 HAs belong to group 1, whereas H3 and H7 HAs belong to group 2 (10). Conventional anti-HA neutralizing antibodies primarily target a few immunodominant epitopes located in proximity to the receptor-binding domain within the globular head region of the molecule (11, 12). Although these antibodies are potentially protective, they are strain-specific because of the high variability of such epitopes, and thus lack, in general, the much-desired broad neutralizing activity. Recently, broadly neutralizing human (13–18) and murine (19) monoclonal antibodies (mAbs) directed against distinct epitopes within the HA stem region have been extensively characterized. These mAbs were shown to interfere with the influenza viruses’ life cycle in different ways (20). By generating monoclonal antibodies from plasmablasts isolated ex vivo, we demonstrated that these broadly neutralizing antibodies could be retrieved from patients infected with or vaccinated against the pandemic H1N1 2009 influenza virus (18, 21). Recent observations that HA stem epitopes are accessible on the majority of HA trimers on intact virions (22), and that a stable HA stem protein that is immunologically intact could be produced (23), provided further hope for the feasibility of a stem-based universal influenza vaccine (24).Notably, HA stem-specific mAbs isolated from humans showed a high degree of affinity maturation, suggesting a memory B-cell origin. These results raised two important questions that we address in the current study. First, what are the baseline levels of broadly cross-reactive stem-binding antibodies and memory B cells? Second, using current influenza vaccines, to what extent can HA stem-specific responses be boosted in comparison with those directed against the HA globular head?Structural studies have clearly demonstrated that the main neutralizing antibody epitopes within the HA stem region are conformation-dependent, and that the integrity of these epitopes requires the presence of the HA1 subunit in addition to the HA2 subunit, which constitute the bulk of the HA stem (16, 17). To be able to directly measure HA stem-reactive antibodies and memory B cells, we used a chimeric HA molecule that expresses the globular head of H9 HA on H1 backbone (25). Our data demonstrate that post-2009 trivalent inactivated vaccines (TIV) induced minimal stem-specific responses in comparison with head-specific responses. On the other hand, immunization with H5N1 generated relatively strong anti-HA stem responses, demonstrating that it is feasible to elicit broadly neutralizing responses in humans given the right immunogen design.     

A recombinant herpes virus expressing influenza hemagglutinin confers protection and induces antibody-dependent cellular cytotoxicity

Despite widespread yearly vaccination, influenza leads to significant morbidity and mortality across the globe. To make a more broadly protective influenza vaccine, it may be necessary to elicit antibodies that can activate effector functions in immune cells, such as antibody-dependent cellular cytotoxicity (ADCC). There is growing evidence supporting the necessity for ADCC in protection against influenza and herpes simplex virus (HSV), among other infectious diseases. An HSV-2 strain lacking the essential glycoprotein D (gD), was used to create ΔgD-2, which is a highly protective vaccine against lethal HSV-1 and HSV-2 infection in mice. It also elicits high levels of IgG2c antibodies that bind FcγRIV, a receptor that activates ADCC. To make an ADCC-eliciting influenza vaccine, we cloned the hemagglutinin (HA) gene from an H1N1 influenza A strain into the ΔgD-2 HSV vector. Vaccination with ΔgD-2::HA_PR8 was protective against homologous influenza challenge and elicited an antibody response against HA that inhibits hemagglutination (HAI⁺), is predominantly IgG2c, strongly activates FcγRIV, and protects against influenza challenge following passive immunization of naïve mice. Prior exposure of mice to HSV-1, HSV-2, or a replication-defective HSV-2 vaccine (dl5-29) does not reduce protection against influenza by ΔgD-2::HA_PR8. This vaccine also continues to elicit protection against both HSV-1 and HSV-2, including high levels of IgG2c antibodies against HSV-2. Mice lacking the interferon-α/β receptor and mice lacking the interferon-γ receptor were also protected against influenza challenge by ΔgD-2::HA_PR8. Our results suggest that ΔgD-2 can be used as a vaccine vector against other pathogens, while also eliciting protective anti-HSV immunity.

Influenza remains a global health threat. Seasonal strains of influenza A and B cause an estimated 5 million cases of severe infections and 500,000 deaths per year (1). Influenza pandemics have caused even greater morbidity and mortality. During the H1N1 pandemic of 1918 to 1919, 500 million people, approximately one-third of the world’s population at that time, were estimated to have been infected with this strain, leading to 50 million deaths (2). The H1N1 pandemic of 2009 is estimated to have caused up to 575,000 deaths (2). Currently, three types of influenza vaccines are offered annually in the United States: a recombinant virus expressing influenza proteins, chemically inactivated virus, and live attenuated virus (3). Regardless of the vaccine type, multiple strains are included to increase the chances of developing sufficient protection against major circulating influenza strains. However, these vaccines primarily elicit a neutralizing antibody response that is sensitive to changes in the influenza virus due to antigenic drift and shift (4). Antigenic drift results from an accumulation of random mutations in influenza antigens, like hemagglutinin (HA), altering sites recognized by the immune system (4). Influenza A strains can also undergo antigenic shift, whereby two different influenza strains infect the same cell to form a reassortant virus with new antigenic properties (4). Due to limited immunity in the population, these new strains are highly virulent, causing widespread epidemics and disease (4). With antigenic drift and shift, vaccine-mediated protection against circulating strains has been insufficient (5). Influenza vaccines that elicit more robust and long-term protection are therefore needed. Notably, if an influenza vaccine with ≥75% efficacy were to be broadly used in the United States, an estimated 19,500 deaths a year could be prevented and direct healthcare costs reduced by $3.5 billion (6).For many years, efforts to improve influenza vaccines have focused on eliciting an immune response for full, broad protection against both circulating and future strains of the virus. These studies have shown that, in general, neutralizing antibodies are sufficient for homologous protection (7). However, achieving heterologous protection may require more broadly neutralizing antibodies or nonneutralizing antibodies able to activate effector immune cells (5). Previous studies have found that passively transferred nonneutralizing monoclonal antibodies can be potently protective in a mouse influenza challenge model (8–10). Several novel strategies have attempted to generate a nonneutralizing response against influenza. For example, vaccines have been created to specifically target the conserved stem region of HA (11–13).Nonneutralizing antibodies stimulate effector cell mechanisms, including antibody-mediated phagocytosis and antibody-dependent cellular cytotoxicity (ADCC), both of which require activation of the Fcγ receptors (FcγRs) (14). Specific isotypes of IgG antibodies are associated with FcγR modulation and subsequent ADCC activation, including the IgG1 and IgG3 subtypes in humans, as well as IgG2a and IgG2c subtypes in mice (15–19). IgG2a and IgG2c isotypes are functionally equivalent and mouse strain-dependent, with IgG2c present in C57BL/6J mice (20). Recent studies have demonstrated that natural infection by influenza and vaccination elicit nonneutralizing antibodies with effector functions that contribute to protection (5, 9, 21–27). In mouse and nonhuman primate challenge models, ADCC-mediating antibodies have demonstrated protection against both homologous and heterologous influenza challenge (9, 28).Recently, we developed a single-cycle herpes simplex virus (HSV) vaccine that completely protects against vaginal, skin, and ocular challenges by HSV-1 and HSV-2 (29, 30). Protection elicited by this vaccine, designated ΔgD-2 for its lack of the essential glycoprotein D (gD) gene, is transferable via passive infusion of immune sera to naïve wild-type mice but not to mice lacking the Fcγ common chain (30). The immune response elicited by ΔgD-2 primarily elicits nonneutralizing antibodies with high levels of FcγRIV-activating function.We asked whether ΔgD-2 could be used as a vaccine platform to induce broadly protective FcγRIV-activating antibodies against a heterologous antigen, such as influenza HA. In this study, we demonstrate that our recombinant vaccine, ΔgD-2::HA_PR8, elicits protection against influenza with a high proportion of FcγRIV-activating antibodies. Additionally, anticipating the use of ΔgD-2 as a vaccine vector against other pathogens, we tested whether our construct would still be protective in mice lacking interferon (IFN) function. Many humans have inborn errors in their IFN signaling pathways, leading to more lethal outcomes in infection (31). Patients with such deficiencies are disproportionately represented among HSV encephalitis cases and are often diagnosed only after presenting with serious symptoms (32–38). This at-risk population underscores the importance of eliciting protection against HSV in the absence of a functional IFN-α/β response. Additionally, many pathogens, such as dengue virus, require mouse models lacking IFN function, and for ease of testing, an efficacious vaccine should remain functional in these mice (39–41). In this study, we demonstrate that ΔgD-2 is a versatile, immunogenic vaccine vector that provides a strong FcγRIV-activating immune response against heterologous pathogens, while maintaining its protective benefit against HSV, in both wild-type and IFN-deficient mice.     

Genetic measurement of memory B-cell recall using antibody repertoire sequencing

Annual influenza vaccinations aim to protect against seasonal infections, and vaccine strain compositions are updated every year. This protection is based on antibodies that are produced by either newly activated or memory B cells recalled from previous encounters with influenza vaccination or infection. The extent to which the B-cell repertoire responds to vaccination and recalls antibodies has so far not been analyzed at a genetic level—which is to say, at the level of antibody sequences. Here, we developed a consensus read sequencing approach that incorporates unique barcode labels on each starting RNA molecule. These labels allow one to combine multiple sequencing reads covering the same RNA molecule to reduce the error rate to a desired level, and they also enable accurate quantification of RNA and isotype levels. We validated this approach and analyzed the differential response of the antibody repertoire to live-attenuated or trivalent-inactivated influenza vaccination. Additionally, we analyzed the antibody repertoire in response to repeated yearly vaccinations with trivalent-inactivated influenza vaccination. We found antibody sequences that were present in both years, providing a direct genetic measurement of B-cell recall.Every year, influenza viruses cause the deaths of an average of 36,000 individuals in the United States alone (1). Although the immunological memory created through vaccination can confer decade-long protection against a particular viral strain, antigenic drift in the original strain and the occurrence of distinct viral strains can enable the virus to evade the immune system (2). As a result, influenza vaccination formulations have to be reevaluated, adjusted, and administered annually to best match the annual influenza strain. Vaccine-induced immunity against influenza is primarily antibody-based, and as such, it relies on the activation of naive B cells or the reactivation (recall) of memory B cells to produce high levels of antibody specific to the vaccine strain. Prior studies approached recall memory responses by measuring plasma antibody levels and specificity or sequencing antibody loci of isolated B cells, with one study concluding that the response to influenza vaccination is pauciclonal (i.e., composed of only a few distinct clones) (3, 4). However, this study and others were limited in the number of B cells that they were able to analyze and not able to show that the same clone recurs during recall. The strength of the recall response, the isotype distribution, and the clonal relationship to others have been unclear.Recently, methods to sequence antibody repertoires of whole organisms and human blood samples were developed and applied to investigate several features of B-cell repertoires (5, 6). This approach has been used to investigate a variety of phenomena, including effects of influenza vaccination, residual disease in leukemia, effects of immune suppression, and differences between memory and naive B-cell compartments (5–11).Analyzing vaccine recall response requires the detection of antibody sequences shared between separate blood samples taken over 12 mo apart. Because of the limited throughput and high error rate of next generation sequencing approaches, it is challenging to query a human blood sample exhaustively and accurately identify these shared sequences. To address these problems, we developed a highly accurate high-throughput approach that relies on the labeling of individual RNA molecules (12–14). We used these labels to generate multiple sequencing reads for each RNA molecule and compose a consensus read for each molecule. First, we validated this approach by sequencing the immunoglobulin heavy chain (IGH) repertoire of a blood sample. We found that this approach was highly accurate, quantitative, and reproducible. Second, we used the consensus read approach to estimate the size of the B-cell repertoire, determining a refined estimate for different B-cell populations. Third, we dissected immune responses to live-attenuated (LAIV) and trivalent-inactivated (TIV) influenza vaccines. LAIV and TIV are known to show distinct immune responses, and we could clearly distinguish the effects of the two vaccine types on the antibody repertoire. Finally, we analyzed the nature of the recall response of individuals to TIV administration in two consecutive years. We found hundreds of unique antibody lineages originating from distinct B-cell memory clones that were activated by vaccination in both consecutive years.     

Initial viral load determines the magnitude of the human CD8 T cell response to yellow fever vaccination

CD8 T cells are a potent tool for eliminating intracellular pathogens and tumor cells. Thus, eliciting robust CD8 T-cell immunity is the basis for many vaccines under development. However, the relationship between antigen load and the magnitude of the CD8 T-cell response is not well-described in a human immune response. Here we address this issue by quantifying viral load and the CD8 T-cell response in a cohort of 80 individuals immunized with the live attenuated yellow fever vaccine (YFV-17D) by sampling peripheral blood at days 0, 1, 2, 3, 5, 7, 9, 11, 14, 30, and 90. When the virus load was below a threshold (peak virus load < 225 genomes per mL, or integrated virus load < 400 genome days per mL), the magnitude of the CD8 T-cell response correlated strongly with the virus load (R² ∼ 0.63). As the virus load increased above this threshold, the magnitude of the CD8 T-cell responses saturated. Recent advances in CD8 T-cell–based vaccines have focused on replication-incompetent or single-cycle vectors. However, these approaches deliver relatively limited amounts of antigen after immunization. Our results highlight the requirement that T-cell–based vaccines should deliver sufficient antigen during the initial period of the immune response to elicit a large number of CD8 T cells that may be needed for protection.CD8 T cells provide a powerful mechanism for elimination of intracellular pathogens and tumor cells. Accordingly, a major thrust of current vaccine research focuses on stimulating robust T-cell immunity for defense against infections such as HIV, malaria, tuberculosis, Ebola virus, herpes viruses, and hepatitis C virus (HCV) (1–8). Inducing effective CD8 T-cell immunity is also an important goal for cancer vaccines (9, 10). However, how antigen load affects the CD8 T-cell response has not been quantified in a detailed manner during a human immune response. In this study we address this question using the human live attenuated yellow fever vaccine (YFV-17D) vaccine.The dynamics of CD8 T-cell responses to intracellular infection have been extensively studied in model systems. Infection typically stimulates a rapid burst of proliferation in antigen-specific CD8 T cells with division occurring as quickly as once in 4–6 h (11). This expansion results in a large population of effector CD8 T cells that aid in clearance of infected cells. Although most (90–95%) of the effector CD8 T cells die, a small fraction differentiate to form long-term memory CD8 T cells (12). Detailed quantitative measurements of the dynamics of virus and the CD8 T-cell response to the YFV-17D vaccine allow us to characterize these basic features of the CD8 T-cell responses in humans. Additionally, tracking the dynamics of both virus and CD8 T cells over time in a large cohort allows us to explore the relationship between amount of antigen and the magnitude of expansion and answer the following questions: Is there a threshold amount of virus required to generate a response? Does the magnitude of the response increase proportionally, or does it saturate with viral load? Although a number of studies have considered the complex relationship between numbers of specific CD8 T cells and virus loads during the chronic phase of HIV and HCV infections (3, 13–23), very few studies (24–27) have investigated these questions in the context of the generation of immune response following acute infections and vaccination.We addressed these questions by measuring the dynamics of both virus and virus-specific CD8 T cells following immunization with the YFV-17D vaccine. The YFV-17D vaccine comprises a highly efficacious, live attenuated virus that causes an acute infection and stimulates a robust immune response conferring lifelong protection against the yellow fever virus (YFV) (28, 29). Because yellow fever is not endemic to the United States, immunization with YFV-17D induces a primary immune response (30, 31). Previous work with YFV-17D has identified CD8 T cells specific for some of the YFV epitopes and defined the stages of expansion, contraction, and memory maintenance (32–38). We now know that YFV stimulates a polyfunctional, broadly targeting, and long-lasting CD8 T-cell response. Of particular note, we have previously demonstrated that the magnitude of the total effector CD8 T-cell response against YFV can be measured using the Ki-67⁺ Bcl-2^lo HLA-DR⁺ CD38⁺ phenotype of activated T cells seen early after vaccination (38). In the current study, we followed a large cohort of 80 individuals with intensive sampling at days 0, 1, 2, 3, 5, 7, 9, 11, 14, 30, and 90 postvaccination to quantify viral load in plasma (39). Additionally, we quantified the magnitude of the YFV-specific effector CD8 T-cell response at days 0, 3, 7, 14, 30, and 90 postvaccination using the Ki-67⁺Bcl-2^lo phenotype. We find that different individuals have different virus loads following infection and generate CD8 T-cell responses of different sizes. This allows us to determine the relationship between virus load and magnitude of the CD8 T-cell response.The majority of vaccines that are currently under development use replication-incompetent or single-cycle vectors such as Modified Vaccinia Ankara, adenovirus, and DNA. Although these approaches are inherently safe, they may express and deliver relatively limited amounts of antigen. Our results emphasize the requirement that T-cell–based vaccines deliver sufficient antigen to elicit a large CD8 T-cell response that may be needed for protection.     

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.     

A common solution to group 2 influenza virus neutralization

The discovery and characterization of broadly neutralizing antibodies (bnAbs) against influenza viruses have raised hopes for the development of monoclonal antibody (mAb)-based immunotherapy and the design of universal influenza vaccines. Only one human bnAb (CR8020) specifically recognizing group 2 influenza A viruses has been previously characterized that binds to a highly conserved epitope at the base of the hemagglutinin (HA) stem and has neutralizing activity against H3, H7, and H10 viruses. Here, we report a second group 2 bnAb, CR8043, which was derived from a different germ-line gene encoding a highly divergent amino acid sequence. CR8043 has in vitro neutralizing activity against H3 and H10 viruses and protects mice against challenge with a lethal dose of H3N2 and H7N7 viruses. The crystal structure and EM reconstructions of the CR8043-H3 HA complex revealed that CR8043 binds to a site similar to the CR8020 epitope but uses an alternative angle of approach and a distinct set of interactions. The identification of another antibody against the group 2 stem epitope suggests that this conserved site of vulnerability has great potential for design of therapeutics and vaccines.Influenza viruses are a significant and persistent threat to human health worldwide. Annual epidemics cause 3–5 million cases of severe illness and up to 0.5 million deaths (1), and periodic influenza pandemics have the potential to kill millions (2). Inhibitors against the viral surface glycoprotein neuraminidase are widely used for the treatment of influenza infections, but their efficacy is being compromised by the emergence of drug-resistant viral strains (3). Vaccination remains the most effective strategy to prevent influenza virus infection. However, protective efficacy is suboptimal in the highest risk groups: infants, the elderly, and the immunocompromised (1). Furthermore, because immunity after vaccination is typically strain-specific and influenza viruses evolve rapidly, vaccines must be updated almost annually. The antigenic composition of the vaccine is based on a prediction of strains likely to circulate in the coming year, therefore, mismatches between vaccine strains and circulating strains occur that can render the vaccine less effective (4). Consequently, there is an urgent need for new prophylactic and therapeutic interventions that provide broad protection against influenza.Immunity against influenza viruses is largely mediated by neutralizing antibodies that target the major surface glycoprotein hemagglutinin (HA) (5, 6). Identification of antigenic sites on HA indicates that influenza antibodies are primarily directed against the immunodominant HA head region (7), which mediates endosomal uptake of the virus into host cells by binding to sialic acid receptors (8). Because of high mutation rates in the HA head region and its tolerance for antigenic changes, antibodies that target the HA head are typically only effective against strains closely related to the strain(s) by which they were elicited, although several receptor binding site-targeting antibodies with greater breadth have been structurally characterized (9–15). In contrast, antibodies that bind to the membrane-proximal HA stem region tend to exhibit much broader neutralizing activity and can target strains within entire subtypes and groups (16–25) as well as across influenza types (24). These stem-directed antibodies inhibit major structural rearrangements in HA that are required for the fusion of viral and host endosomal membranes and thus, prevent the release of viral contents into the cell (8). The stem region is less permissive for mutations than the head and relatively well-conserved across divergent influenza subtypes.Anti-stem antibodies are elicited in some, but not all, individuals during influenza infection or vaccination (20, 26) and thus, hold great promise as potential broad spectrum prophylactic or therapeutic agents and for the development of a universal influenza vaccine (27–29). The majority of the known heterosubtypic stem binding antibodies neutralize influenza A virus subtypes belonging to group 1 (17–20, 23, 25). Furthermore, two antibodies that target a similar epitope in the HA stem, like most heterosubtypic group 1 antibodies, are able to more broadly recognize both group 1 and 2 influenza A viruses (22) or influenza A and B viruses (24). Strikingly, group 2-specific broadly neutralizing Abs (bnAbs) seem to be rare, because only one has been reported to date (21). CR8020 uniquely targets a distinct epitope in the stem in close proximity to the viral membrane at the HA base and binds lower down the stem than any other influenza HA antibody (21).In the discovery process that led to the isolation of bnAb CR8020, we recovered additional group 2-specific bnAbs. Here, we describe one such bnAb, CR8043, which recognizes a similar but nonidentical footprint on the HA as CR8020 and approaches the HA from a different angle. Furthermore, these two bnAbs are derived from different germ-line genes and, consequently, use distinct sets of interactions for HA recognition. Thus, the human immune system is able to recognize this highly conserved epitope in different ways using different germ-line genes. Hence, this valuable information can be used for the design of therapeutics and vaccines targeting this site of vulnerability in group 2 influenza A viruses that include the pandemic H3N2 subtype.     

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.     

Relationship of the quaternary structure of human secretory IgA to neutralization of influenza virus

Secretory IgA (S-IgA) antibodies, the major contributors to humoral mucosal immunity to influenza virus infection, are polymeric Igs present in many external secretions. In the present study, the quaternary structures of human S-IgA induced in nasal mucosa after administration of intranasal inactivated influenza vaccines were characterized in relation to neutralization potency against influenza A viruses. Human nasal IgA antibodies have been shown to contain at least five quaternary structures. Direct and real-time visualization of S-IgA using high-speed atomic force microscopy (AFM) demonstrated that trimeric and tetrameric S-IgA had six and eight antigen-binding sites, respectively, and that these structures exhibited large-scale asynchronous conformational changes while capturing influenza HA antigens in solution. Furthermore, trimeric, tetrameric, and larger polymeric structures, which are minor fractions in human nasal IgA, displayed increased neutralizing potency against influenza A viruses compared with dimeric S-IgA, suggesting that the larger polymeric than dimeric forms of S-IgA play some important roles in protection against influenza A virus infection in the human upper respiratory tract.Antibodies in respiratory mucosa are primary mediators of protective immunity against influenza. Notably, preexisting secretory IgA (S-IgA) antibodies can provide immediate immunity by eliminating a pathogen before the virus passes the mucosal barrier (1–3). Parenteral vaccination induces serum IgG but not S-IgA, so vaccine efficacy is limited. In contrast, intranasal administration of an inactivated influenza vaccine elicits both S-IgA and IgG responses, thus improving the protective efficacy of current vaccination procedures (4–8).IgA in human serum exists predominantly in the form of monomers, whereas the majority of IgA in external secretions is present in the form of polymers. These polymeric IgA forms are associated with the extracellular portion of the polymeric Ig receptor, generating a complex (receptor + polymeric IgA) called S-IgA (9). S-IgA primarily corresponds to dimeric IgA, although low levels of some larger polymeric forms, particularly tetramers, are also present (9–15). Polymeric S-IgA has been shown (both in vitro and in experimental animal models) to be more effective than monomeric IgA or IgG for the neutralization of influenza viruses (16–19). However, little is known of the quaternary structures and neutralizing potencies in viral infection of the various forms of polymeric S-IgA in the human nasal mucosa. In this study, the quaternary structures and neutralizing potencies of nasal antibodies against influenza virus were examined using nasal wash samples from healthy adults who had received intranasally administered inactivated influenza vaccines. These nasal wash samples, containing variously sized Igs, were separated by gel filtration chromatography (GFC) and assessed for neutralization activity against influenza virus. The quaternary structures of the nasal IgA induced by intranasally administered inactivated influenza vaccines then were determined using biochemical techniques and high-speed atomic force microscopy (AFM). We found that human nasal IgA comprised at least five quaternary structures, including monomer, dimer, trimer, and tetramer structures, as well as a polymeric form larger than the tetramer structure. Among these forms, the polymeric structure demonstrated higher neutralizing potency against seasonal influenza viruses (H3N2) and highly pathogenic avian influenza virus (H5N1) compared with the dimeric form, suggesting that large polymeric S-IgA antibodies play crucial roles in protective immunity against influenza virus infection of the human upper respiratory tract.     

Vaccination of monoglycosylated hemagglutinin induces cross-strain protection against influenza virus infections

The 2009 H1N1 pandemic and recent human cases of H5N1, H7N9, and H6N1 in Asia highlight the need for a universal influenza vaccine that can provide cross-strain or even cross-subtype protection. Here, we show that recombinant monoglycosylated hemagglutinin (HA_mg) with an intact protein structure from either seasonal or pandemic H1N1 can be used as a vaccine for cross-strain protection against various H1N1 viruses in circulation from 1933 to 2009 in mice and ferrets. In the HA_mg vaccine, highly conserved sequences that were originally covered by glycans in the fully glycosylated HA (HA_fg) are exposed and thus, are better engulfed by dendritic cells (DCs), stimulated better DC maturation, and induced more CD8+ memory T cells and IgG-secreting plasma cells. Single B-cell RT-PCR followed by sequence analysis revealed that the HA_mg vaccine activated more diverse B-cell repertoires than the HA_fg vaccine and produced antibodies with cross-strain binding ability. In summary, the HA_mg vaccine elicits cross-strain immune responses that may mitigate the current need for yearly reformulation of strain-specific inactivated vaccines. This strategy may also map a new direction for universal vaccine design.HA glycoprotein on the surface of influenza virus is a major target for infectivity-neutralizing antibodies. However, the antigenic drift and shift of this protein mean that influenza vaccines must be reformulated annually to include HA proteins of the viral strains predicted for the upcoming flu season (1). This time-consuming annual reconfiguration process has led to efforts to develop new strategies and identify conserved epitopes recognized by broadly neutralizing antibodies as the basis for designing universal vaccines to elicit antibodies with a broad protection against various strains of influenza infection (2–6). Previous studies have shown that the stem region of HA is more conserved and able to induce cross-reactive and broadly neutralizing antibodies (7–9) to prevent the critical fusion of viral and endosomal membranes in the influenza lifecycle (10–14). Other broadly neutralizing antibodies have been found to bind regions near the receptor binding site of the globular domain, although these antibodies are fewer in number (15, 16).Posttranslational glycosylation of HA plays an important role in the lifecycle of the influenza virus and also contributes to the structural integrity of HA and the poor immune response of the infected hosts. Previously, we trimmed down the size of glycans on avian influenza H5N1 HA with enzymes and showed that H5N1 HA with a single N-linked GlcNAc at each glycosylation site [monoglycosylated HA (HA_mg)] produces a superior vaccine with more enhanced antibody response and neutralization activity against the homologous influenza virus than the fully glycosylated HA (HA_fg) (17). Here, to test whether the removal of glycans from HA contributes to better immune responses and possibly protects against heterologous strains of influenza viruses, we compared and evaluated the efficacy of HA glycoproteins with various lengths of glycans as potential vaccine candidates.     

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.     

Effective and lesion-free cutaneous influenza vaccination

The current study details efficient lesion-free cutaneous vaccination via vaccine delivery into an array of micropores in the skin, instead of bolus injection at a single site. Such delivery effectively segregated vaccine-induced inflammation, resulting in rapid resolution of the inflammation, provided that distances between any two micropores were sufficient. When the inoculation site was treated by FDA-approved nonablative fractional laser (NAFL) before insertion of a PR8 model influenza vaccine-packaged, biodegradable microneedle array (MNs), mice displayed vigorous antigen-uptake, eliciting strong Th1-biased immunity. These animals were completely protected from homologous viral challenges, and fully or partially protected from heterologous H1N1 and H3N2 viral challenges, whereas mice receiving MNs alone suffered from severe illnesses or died of similar viral challenges. NAFL-mediated adjuvanicity was ascribed primarily to dsDNA and other “danger” signals released from laser-damaged skin cells. Thus, mice deficient in dsDNA-sensing pathway, but not Toll like receptor (TLR) or inflammasome pathways, showed poor responses to NAFL. Importantly, with this novel approach both mice and swine exhibited strong protective immunity without incurring any appreciable skin irritation, in sharp contrast to the overt skin irritation caused by intradermal injections. The effective lesion-free cutaneous vaccination merits further clinical studies.Substantial evidence has shown that skin is a more potent site for vaccination than muscle because the former contains a large number of antigen presenting cells (APCs) and abundant network of lymphatic vessels. Moreover, skin offers potential for painless, needle-free, and self-applicable immunization, which would particularly benefit annual influenza vaccination of large populations (1, 2). However, skin immunization has not been broadly adopted to date, due to lack of safe adjuvants and technical difficulties in injecting vaccines into the ultrathin (<2 mm) skin tissue (3, 4). We have demonstrated that controllable skin injury can serve as safe “adjuvant” for cutaneous vaccination and similar observations have been also made by other investigators (5–7). Treatment of the inoculation site with nonablative fractional laser (NAFL) generates an array of microthermal zones (MTZs) beneath the stratum corneum (8). The dying cells in the MTZs release “danger” signals that provoke sterile inflammation that is however constrained within individual MTZs. This array of microsterile inflammatory zones is resolved quickly, effectively averting skin lesion, provided that affected and unaffected areas of the skin are adequately balanced (8). Importantly, despite fast resolution, the microinflammation zones prove sufficient in augmentation of adaptive immune responses against the vaccine (5). In addition, this standalone adjuvant does not affect the volume of administration or formulation of the vaccine, which are serious hurdles for skin immunization because only a limited volume and nonviscous vaccines are suitable for skin injection (9). In this regard, emulsion-based adjuvants like Alum and MF59 are excluded from a use as cutaneous adjuvant owing to strong and persistent skin reactions induced by the adjuvants (3, 4, 10).Apart from adjuvants, intradermal (ID) delivery of influenza vaccine not only is challenging but also provokes a high rate of pain and skin irritation compared with intramuscular (IM) immunization. In the last decade, a variety of strategies have been explored to deliver vaccines into the skin, among which microneedle has received considerable attentions (11). The technology uses single or an array of ultra-short and ultra-thin needles to penetrate into the skin for vaccine delivery. These microneedles are long enough to cross the stratum corneum barrier, but short enough to avoid pain, offering a method for minimally invasive and painless vaccine delivery. Notably, an intradermal microinjection system has already been approved for delivering a reduced dose (9 μg) of seasonal influenza vaccine in adults in USA and Europe and a typical dose (15 μg) in the elderly in Europe. The thinner and shorter microneedle (1.5 mm in length) reduces pain significantly. The pain may be further reduced by a microneedle array (MNs) or a patch where each array contains hundreds or thousands of tiny microneedles (12–15). To load a sufficient amount of vaccine in a patch, these microneedles are usually densely packed in a small patch (∼1 cm²). Nevertheless, these highly dense microneedle arrays hardly address skin reactogenicity in relevant animal models.To robustly reduce or completely eliminate skin lesion, we expanded the microfractional concept from adjuvant to vaccine delivery in the current study. We fabricated an array of biodegradable microneedles with sufficient distances between individual microneedles to constrain vaccine-induced inflammation. Prevention of vaccine-induced inflammation from spreading into nearby micropores warranted rapid resolution of inflammation as well as minimal skin lesion. Moreover, when influenza vaccine-packaged MNs was applied into a NAFL-treated site, the vaccine induced much broader immunity against homo- and heterologous strains of influenza viruses than the array alone in mice. The combination also gave rise to strong protective immunity but with little skin lesion in swine, an animal model with skin resembling that of humans.     

A 680,000-person megastudy of nudges to encourage vaccination in pharmacies

Encouraging vaccination is a pressing policy problem. To assess whether text-based reminders can encourage pharmacy vaccination and what kinds of messages work best, we conducted a megastudy. We randomly assigned 689,693 Walmart pharmacy patients to receive one of 22 different text reminders using a variety of different behavioral science principles to nudge flu vaccination or to a business-as-usual control condition that received no messages. We found that the reminder texts that we tested increased pharmacy vaccination rates by an average of 2.0 percentage points, or 6.8%, over a 3-mo follow-up period. The most-effective messages reminded patients that a flu shot was waiting for them and delivered reminders on multiple days. The top-performing intervention included two texts delivered 3 d apart and communicated to patients that a vaccine was “waiting for you.” Neither experts nor lay people anticipated that this would be the best-performing treatment, underscoring the value of simultaneously testing many different nudges in a highly powered megastudy.

Encouraging vaccination has emerged as a pressing policy problem during the COVID-19 crisis. Although COVID-19 vaccines were widely available to Americans beginning in the late spring of 2021, the United States did not achieve President Biden’s goal of 70% of American adults receiving their first dose by July 1 (1). Further, millions who received a first dose of a COVID-19 vaccine failed to receive a second dose on schedule (1). States, cities, and the federal government have been left scrambling to find cost-effective ways to motivate vaccine uptake (2, 3).How can policymakers more-effectively encourage vaccination? Past research suggests that simple, low-cost nudges can help (4–8). For instance, when doctors’ offices and health systems send text-based reminders to patients that vaccines are available or reserved for them, this significantly boosts both flu vaccination (8) and COVID-19 vaccination rates (9).Increasing compliance with public health recommendations is not just a persuasion problem. Decades of research show that many people fail to follow through on their intentions when it comes to decisions about their health (10). In the context of vaccination, one study found that only 79% of those who intended to get a flu shot actually followed through (11). Follow-through failures are driven by a combination of forgetting, failing to anticipate and plan for obstacles, and low motivation (10, 12). Psychologically informed reminders have the potential to bridge some of these “intention–action” gaps (13), and pharmacies have become a front line for providing vaccinations (14), providing 37% of flu shots in the United States as of 2020 (15). As a result, understanding what reminders work in this context is particularly important, especially because patient demographics differ in pharmacies and doctors’ offices (16).Although there are many important differences between COVID-19 and influenza, both are respiratory ailments for which the risk of infection and severe illness can be reduced with safe, widely available vaccines. In the fall of 2020, anticipating the pressing need for insights about behaviorally informed COVID-19 vaccination messaging, we partnered with Walmart pharmacies to conduct a megastudy (17) of text-based reminder messages to encourage flu vaccination.Pharmacy patients (n = 689,693) were randomly assigned to receive one of 22 different text message reminders or to a business-as-usual control condition with no messages. Text messages were developed by separate teams of behavioral scientists and used a variety of different tactics to nudge vaccination.While all 22 interventions were designed to encourage inoculation against the flu, scientists were asked to develop interventions that they believed would be reusable to encourage COVID-19 vaccination. The resulting interventions relied on a wide range of behavioral insights and varied both in their content and timing of delivery.To assess how well the relative success of these messages could be forecasted ex ante, both the scientists who developed the texts and a separate sample of lay survey respondents predicted the impact of different interventions on flu vaccination rates.     

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.     

Interprotomer disulfide-stabilized variants of the human metapneumovirus fusion glycoprotein induce high titer-neutralizing responses

Human metapneumovirus (HMPV) is a major cause of respiratory disease worldwide, particularly among children and the elderly. Although there is no licensed HMPV vaccine, promising candidates have been identified for related pneumoviruses based on the structure-based stabilization of the fusion (F) glycoprotein trimer, with prefusion-stabilized F glycoprotein trimers eliciting significantly higher neutralizing responses than their postfusion F counterparts. However, immunization with HMPV F trimers in either prefusion or postfusion conformations has been reported to elicit equivalent neutralization responses. Here we investigate the impact of stabilizing disulfides, especially interprotomer disulfides (IP-DSs) linking protomers of the F trimer, on the elicitation of HMPV-neutralizing responses. We designed F trimer disulfides, screened for their expression, and used electron microscopy (EM) to confirm their formation, including that of an unexpected postfusion variant. In mice, IP-DS–stabilized prefusion and postfusion HMPV F elicited significantly higher neutralizing responses than non–IP-DS–stabilized HMPV Fs. In macaques, the impact of IP-DS stabilization was more measured, although IP-DS–stabilized variants of either prefusion or postfusion HMPV F induced neutralizing responses many times the average titers observed in a healthy human cohort. Serological and absorption-based analyses of macaque responses revealed elicited HMPV-neutralizing responses to be absorbed differently by IP-DS–containing and by non–IP-DS–containing postfusion Fs, suggesting IP-DS stabilization to alter not only the immunogenicity of select epitopes but their antigenicity as well. We speculate the observed increase in immunogenicity by IP-DS trimers to be related to reduced interprotomer flexibility within the HMPV F trimer.

Human metapneumovirus (HMPV) is a globally widespread respiratory pathogen (1–13) primarily affecting infants, the elderly, and those that are immune compromised (reviewed in ref. 14). Disease symptoms are similar to those of the closely related respiratory syncytial virus (RSV) (15), with rates of hospitalization in older adults approaching those of influenza (6). Phylogenetic analysis (SI Appendix, Fig. S1) shows HMPV to comprise two related subtypes, A and B, which are closely related to avian metapneumoviruses, from which HMPV might have evolved (1, 16, 17).There is currently no licensed vaccine or treatment for HMPV (2–4, 18). Recently, promising vaccine candidates for RSV as well as for parainfluenza viruses types 1 through 4 have been developed using structure-based vaccine design, in which prefusion-stabilized versions of the fusion (F) glycoprotein have been used to induce high titer-neutralizing responses; this approach has succeeded in animal models (19–23) and for RSV in recent human clinical trials (24). Unfortunately, prefusion F-based stabilization with the HMPV F glycoprotein does not induce improved neutralization titers compared to postfusion HMPV F (18, 25, 26).In the absence of a difference in titers induced by prefusion versus postfusion forms of HMPV F, we explored whether there was a structure-based vaccine approach that might increase neutralizing titers elicited by HMPV F. With both human and bovine RSV F, stabilization in the prefusion conformation with multiple disulfide bonds significantly increased neutralizing titers (27, 28). The increase was especially evident with interprotomer disulfides (IP-DSs), covalently cross-linking together protomers within the trimer (27). We therefore sought to test the impact of disulfides—and especially IP-DSs—on the elicitation of HMPV-neutralizing responses. We evaluated the prefusion HMPV F structure (PDB ID 5WB0) (26) for sites suitable for the introduction of either intraprotomer- or IP-DS–bonding mutations, which we then synthesized, expressed, and tested antigenically. We determined cryoEM structures to delineate F conformation and atomic-level details of stabilization and assessed immunogenicity in mice and rhesus macaques. Overall, IP-DSs—of both prefusion and postfusion F—induced significantly higher HMPV-neutralizing responses than non–IP-DS–stabilized variants, suggesting that in addition to fixing a particular conformation, IP-DSs appear to be capable of enhancing the immunogenicity of neutralizing responses.