Intranasal inoculation of an MVA-based vaccine induces IgA and protects the respiratory tract of hACE2 mice from SARS-CoV-2 infection

Current vaccines have greatly diminished the severity of the COVID-19 pandemic, even though they do not entirely prevent infection and transmission, likely due to insufficient immunity in the upper respiratory tract. Here, we compare intramuscular and intranasal administration of a live, replication-deficient modified vaccinia virus Ankara (MVA)–based Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) spike (S) vaccine to raise protective immune responses in the K18-hACE2 mouse model. Using a recombinant MVA expressing firefly luciferase for tracking, live imaging revealed luminescence of the respiratory tract of mice within 6 h and persisting for 3 d following intranasal inoculation, whereas luminescence remained at the site of intramuscular vaccination. Intramuscular vaccination induced S-binding–Immunoglobulin G (IgG) and neutralizing antibodies in the lungs, whereas intranasal vaccination also induced Immunoglobulin A (IgA) and higher levels of antigen-specific CD3⁺CD8⁺IFN-γ⁺ T cells. Similarly, IgG and neutralizing antibodies were present in the blood of mice immunized intranasally and intramuscularly, but IgA was detected only after intranasal inoculation. Intranasal boosting increased IgA after intranasal or intramuscular priming. While intramuscular vaccination prevented morbidity and cleared SARS-CoV-2 from the respiratory tract within several days after challenge, intranasal vaccination was more effective as neither infectious virus nor viral messenger (m)RNAs were detected in the nasal turbinates or lungs as early as 2 d after challenge, indicating prevention or rapid elimination of SARS-CoV-2 infection. Additionally, we determined that neutralizing antibody persisted for more than 6 mo and that serum induced to the Wuhan S protein neutralized pseudoviruses expressing the S proteins of variants, although with less potency, particularly for Beta and Omicron.

The rapid development of SARS-CoV-2 vaccines was a stunning achievement that is contributing to the control of the COVID-19 pandemic. Several types of vaccines—including mRNA, adenovirus-vectors, recombinant spike (S) protein, and inactivated Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)—have demonstrated the ability to protect against severe disease. Nevertheless, these vaccines, which are administered systemically, reduce but do not prevent virus infection and transmission, and therefore approaches that provide further immunity are desirable (1). SARS-CoV-2 spreads by droplet and aerosol so that the nasal and oral mucosa are the first barriers to infection. In general, the intranasal (IN) route of vaccination induces greater mucosal immunity compared with the intramuscular (IM) route. An example is the live, attenuated influenza virus vaccine, called LAIV or FluMist, which is approved as a nasal spray in some countries. Unlike inactivated influenza vaccine, LAIV induces nasal Immunoglobulin A (IgA) and CD8⁺ T cells (2). Similarly, IN administration of adenovirus-vectored SARS-CoV-2 vaccines reduce viral loads in upper and lower respiratory tracts following challenge in several animal models (3–6) and an aerosolized vaccine appeared safe and immunogenic in a phase I trial (7), although a trial of another adenovirus-based nasal spray vaccine was discontinued because of low immunogenicity (https://ir.altimmune.com/news-releases/news-release-details/altimmune-announces-update-adcovidtm-phase-1-clinical-trial). Studies of IN vaccination with additional vectors are needed.Modified vaccinia virus Ankara (MVA) is a highly attenuated, replication-defective, immunogenic smallpox vaccine strain that is undergoing clinical testing as a vector for multiple pathogens (8) as well as SARS-CoV-2 (www.clinical trials.gov). Although usually administered IM or subcutaneously, several reports have shown that MVA-based vectors induce protective mucosal and systemic immune responses when administered IN to animals (9–13). In addition, combined IM and IN vaccination of camels with an MVA-based vaccine reduced excretion of Middle East respiratory syndrome (MERS)-CoV, although the efficacy of IN alone was not reported (14).The present study was initiated to extend previous demonstrations of the ability of IM administered MVA-vectored vaccines to protect against SARS-CoV-2 challenge in animal models (15–18). We previously reported (15) that IM injection of MVA expressing a modified S protein with mutations that stabilized the prefusion form, inactivated the furin cleavage site, and deleted the endoplasmic retention signal induced a type 1 immune response with neutralizing antibody and CD8⁺IFN-γ⁺ T cells, and protected K18-hACE2 transgenic mice from respiratory infection with SARS-CoV-2. In addition, passive transfer of serum from vaccinated mice to unvaccinated mice protected them from lethal SARS-CoV-2 infection. Here, we show persistence of neutralizing antibody and protection of transgenic hACE2 mice for more than 6 mo after one or two IM inoculations with an MVA-based modified S protein vaccine. However, whereas IM vaccination induced Immunoglobulin G (IgG) neutralizing antibodies and cleared infection of the respiratory tract, IN inoculations also induced IgA antibodies in the lungs and blood, and after two IN vaccinations neither SARS-CoV-2 nor subgenomic (sg) mRNAs were detected in the nasal turbinates or lungs at 2 or 5 d after challenge. IN delivery of a live recombinant vaccine has the potential to reduce infection and transmission of SARS-CoV-2.     

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.     

Modeling SARS-CoV-2 Infection in Mice Using Lentiviral hACE2 Vectors Infers Two Modes of Immune Responses to SARS-CoV-2 Infection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a severe global pandemic. Mice models are essential to investigate infection pathology, antiviral drugs, and vaccine development. However, wild-type mice lack the human angiotensin-converting enzyme 2 (hACE2) that mediates SARS-CoV-2 entry into human cells and consequently are not susceptible to SARS-CoV-2 infection. hACE2 transgenic mice could provide an efficient COVID-19 model, but are not always readily available, and practically restricted to specific strains. Therefore, there is a dearth of additional mouse models for SARS-CoV-2 infection. We applied lentiviral vectors to generate hACE2 expression in interferon receptor knock-out (IFNAR1^−/−) mice. Lenti-hACE2 transduction supported SARS-CoV-2 replication in vivo, simulating mild acute lung disease. Gene expression analysis revealed two modes of immune responses to SARS-CoV-2 infection: one in response to the exposure of mouse lungs to SARS-CoV-2 particles in the absence of productive viral replication, and the second in response to productive SARS-CoV-2 infection. Our results infer that immune response to immunogenic elements on incoming virus or in productively infected cells stimulate diverse immune effectors, even in absence of type I IFN signaling. Our findings should contribute to a better understanding of the immune response triggered by SARS-CoV-2 and to further elucidate COVID-19.     

Prior SARS-CoV-2 infection and COVID-19 vaccine effectiveness against outpatient illness during widespread circulation of SARS-CoV-2 Omicron variant,US Flu VE network

Background

We estimated combined protection conferred by prior SARS-CoV-2 infection and COVID-19 vaccination against COVID-19-associated acute respiratory illness (ARI).

Methods

During SARS-CoV-2 Delta (B.1.617.2) and Omicron (B.1.1.529) variant circulation between October 2021 and April 2022, prospectively enrolled adult patients with outpatient ARI had respiratory and filter paper blood specimens collected for SARS-CoV-2 molecular testing and serology. Dried blood spots were tested for immunoglobulin-G antibodies against SARS-CoV-2 nucleocapsid (NP) and spike protein receptor binding domain antigen using a validated multiplex bead assay. Evidence of prior SARS-CoV-2 infection also included documented or self-reported laboratory-confirmed COVID-19. We used documented COVID-19 vaccination status to estimate vaccine effectiveness (VE) by multivariable logistic regression by prior infection status.

Results

Four hundred fifty-five (29%) of 1577 participants tested positive for SARS-CoV-2 infection at enrollment; 209 (46%) case-patients and 637 (57%) test-negative patients were NP seropositive, had documented previous laboratory-confirmed COVID-19, or self-reported prior infection. Among previously uninfected patients, three-dose VE was 97% (95% confidence interval [CI], 60%–99%) against Delta, but not statistically significant against Omicron. Among previously infected patients, three-dose VE was 57% (CI, 20%–76%) against Omicron; VE against Delta could not be estimated.

Conclusions

Three mRNA COVID-19 vaccine doses provided additional protection against SARS-CoV-2 Omicron variant-associated illness among previously infected participants.     

Proper Selection of In Vitro Cell Model Affects the Characterization of the Neutralizing Antibody Response against SARS-CoV-2

(1) Background: Our aim is the evaluation of the neutralizing activity of BNT162b2 mRNA vaccine-induced antibodies in different in vitro cellular models, as this still represents one of the surrogates of protection against SARS-CoV-2 viral variants. (2) Methods: The entry mechanisms of SARS-CoV-2 in three cell lines (Vero E6, Vero E6/TMPRSS2 and Calu-3) were evaluated with both pseudoviruses and whole virus particles. The neutralizing capability of sera collected from vaccinated subjects was characterized through cytopathic effects and Real-Time RT PCR. (3) Results: In contrast to Vero E6 and Vero E6/TMPRSS2, Calu-3 allowed the evaluation of both viral entry mechanisms, resembling what occurs during natural infection. The choice of an appropriate cellular model can decisively influence the determination of the neutralizing activity of antibodies against SARS-CoV-2 variants. Indeed, the lack of correlation between neutralizing data in Calu-3 and Vero E6 demonstrated that testing the antibody inhibitory activity by using a single cell model possibly results in an inaccurate characterization. (4) Conclusions: Cellular systems allowing only one of the two viral entry pathways may not fully reflect the neutralizing activity of vaccine-induced antibodies moving increasingly further away from possible correlates of protection from SARS-CoV-2 infection.     

Structural Analysis of Neutralizing Epitopes of the SARS-CoV-2 Spike to Guide Therapy and Vaccine Design Strategies

Coronavirus research has gained tremendous attention because of the COVID-19 pandemic, caused by the novel severe acute respiratory syndrome coronavirus (nCoV or SARS-CoV-2). In this review, we highlight recent studies that provide atomic-resolution structural details important for the development of monoclonal antibodies (mAbs) that can be used therapeutically and prophylactically and for vaccines against SARS-CoV-2. Structural studies with SARS-CoV-2 neutralizing mAbs have revealed a diverse set of binding modes on the spike’s receptor-binding domain and N-terminal domain and highlight alternative targets on the spike. We consider this structural work together with mAb effects in vivo to suggest correlations between structure and clinical applications. We also place mAbs against severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses in the context of the SARS-CoV-2 spike to suggest features that may be desirable to design mAbs or vaccines capable of conferring broad protection.     

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

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

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

SARS-CoV-2 Virus-like Particles Produced by a Single Recombinant Baculovirus Generate Anti-S Antibody and Protect against Variant Challenge

Coronavirus Disease 2019 (COVID-19), caused by infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has highlighted the need for the rapid generation of efficient vaccines for emerging disease. Virus-like particles, VLPs, are an established vaccine technology that produces virus-like mimics, based on expression of the structural proteins of a target virus. SARS-CoV-2 is a coronavirus where the basis of VLP formation has been shown to be the co-expression of the spike, membrane and envelope structural proteins. Here we describe the generation of SARS-CoV-2 VLPs by the co-expression of the salient structural proteins in insect cells using the established baculovirus expression system. VLPs were heterologous ~100 nm diameter enveloped particles with a distinct fringe that reacted strongly with SARS-CoV-2 convalescent sera. In a Syrian hamster challenge model, non-adjuvanted VLPs induced neutralizing antibodies to the VLP-associated Wuhan S protein and reduced virus shedding and protected against disease associated weight loss following a virulent challenge with SARS-CoV-2 (B.1.1.7 variant). Immunized animals showed reduced lung pathology and lower challenge virus replication than the non-immunized controls. Our data suggest SARS-CoV-2 VLPs offer an efficient vaccine that mitigates against virus load and prevents severe disease.     

Third Early “Booster” Dose Strategy in France of bnt162b2 SARS-CoV-2 Vaccine in Allogeneic Hematopoietic Stem Cell Transplant Recipients Enhances Neutralizing Antibody Responses

Immunocompromised individuals generally fail to mount efficacious immune humoral responses following vaccination. The emergence of SARS-CoV-2 variants of concern has raised the question as to whether levels of anti-spike protein antibodies achieved after two or three doses of the vaccine efficiently protect against breakthrough infection in the context of immune suppression. We used a fluorescence-based neutralization assay to test the sensitivity of SARS-CoV-2 variants (ancestral variant, Beta, Delta, and Omicron BA.1) to the neutralizing response induced by vaccination in highly immunosuppressed allogeneic HSCT recipients, tested after two and three doses of the BNT162b2 vaccine. We show that neutralizing antibody responses to the Beta and Delta variants in most immunocompromised HSCT recipients increased after three vaccine doses up to values similar to those observed in twice-vaccinated healthy adults and were significantly lower against Omicron BA.1. Overall, neutralization titers correlated with the amount of anti-S-RBD antibodies measured by means of enzyme immunoassay, indicating that commercially available assays can be used to quantify the anti-S-RBD antibody response as a reliable surrogate marker of humoral immune protection in both immunocompetent and immunocompromised individuals. Our findings support the recommendation of additional early vaccine doses as a booster of humoral neutralizing activity against emerging variants, in HSCT immunocompromised patients. In the context of Omicron circulation, it further emphasizes the need for reinforcement of preventive measures including the administration of monoclonal antibodies in this high-risk population.     

Presence of SARS-CoV-2 RNA on Surfaces of Public Places and a Transportation System Located in a Densely Populated Urban Area in South America

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible RNA virus that causes COVID-19. Being aware of the presence of the virus on different types of surfaces and in different environments, and having a protocol for its detection, is important to understand the dynamics of the virus and its shedding patterns. In Ecuador, the detection of viral RNA in urban environmental samples has not been a priority. The present study analyzed samples from two densely populated neighborhoods and one public transportation system in Quito, Ecuador. Viral RNA presence was assessed using RT-LAMP. Twenty-eight out of 300 surfaces tested positive for SARS-CoV-2 RNA (9.33%). Frequently touched surfaces, especially in indoor spaces and on public transportation, were most likely to be positive for viral RNA. Positivity rate association for the two neighborhoods and for the surface type was not found. This study found viral RNA presence on urban surfaces; this information provides an insight into viral dissemination dynamics. Monitoring environmental SARS-CoV-2 could support the public health prevention strategies in Quito, Ecuador.     

慢性HBV感染者接种新型冠状病毒疫苗的安全性及有效性研究

目的?分析慢性HBV感染者全程接种3剂新型冠状病毒（severe acute respiratory syndrome coronavirus 2, SARS-CoV-2）疫苗的安全性及有效性。方法?纳入2021年10月—2022年8月在解放军总医院第五医学中心就诊并自愿接种SARS-CoV-2疫苗的慢性HBV感染者107例，在第0（基线）、1、2、4、7、8月时进行访视并定期检测血常规、血生化及凝血四项等实验室指标。通过记录慢性HBV感染者接种每剂SARS-CoV-2疫苗后28 d内的不良反应及实验室指标的动态变化特点以评估其安全性。在随访时留取血浆样本检测中和抗体滴度以评估其有效性，同时分析抗体滴度可能的影响因素。结果?慢性HBV感染者接种3剂SARS-CoV-2疫苗后不良反应发生率依次为22.43%（24/107）、19.79%（19/96）和16.67%（12/72），最常见的局部不良反应为接种部位疼痛，最常见的全身性的不良反应为疲劳、乏力；接种疫苗后生化学及病毒学指标未出现恶化；接种每剂SARS-CoV-2疫苗后中和抗体滴度均显著上升（P均＜0.05），而后随着时间逐渐下降；抗体阳性组受试者年龄低于阴性组受试者[（42.27±9.40）岁 vs. 48.00（43.00，53.50）岁，P = 0.040]。结论?慢性HBV感染者接种SARS-CoV-2疫苗后具有良好的安全性及有效性，不良反应发生率低且未发生疫苗相关严重不良事件；接种第3剂加强针后产生的中和抗体反应最为强烈；而年龄则会影响慢性HBV感染者接种疫苗后的抗体滴度。     

Elicitation of Broadly Neutralizing Antibodies against B.1.1.7, B.1.351, and B.1.617.1 SARS-CoV-2 Variants by Three Prototype Strain-Derived Recombinant Protein Vaccines

The ongoing coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Most of the currently approved SARS-CoV-2 vaccines use the prototype strain-derived spike (S) protein or its receptor-binding domain (RBD) as the vaccine antigen. The emergence of several novel SARS-CoV-2 variants has raised concerns about potential immune escape. In this study, we performed an immunogenicity comparison of prototype strain-derived RBD, S1, and S ectodomain trimer (S-trimer) antigens and evaluated their induction of neutralizing antibodies against three circulating SARS-CoV-2 variants, including B.1.1.7, B.1.351, and B.1.617.1. We found that, at the same antigen dose, the RBD and S-trimer vaccines were more potent than the S1 vaccine in eliciting long-lasting, high-titer broadly neutralizing antibodies in mice. The RBD immune sera remained highly effective against the B.1.1.7, B.1.351, and B.1.617.1 variants despite the corresponding neutralizing titers decreasing by 1.2-, 2.8-, and 3.5-fold relative to that against the wild-type strain. Significantly, the S-trimer immune sera exhibited comparable neutralization potency (less than twofold variation in neutralizing GMTs) towards the prototype strain and all three variants tested. These findings provide valuable information for further development of recombinant protein-based SARS-CoV-2 vaccines and support the continued use of currently approved SARS-CoV-2 vaccines in the regions/countries where variant viruses circulate.     

Neuroinvasion and Neurotropism by SARS-CoV-2 Variants in the K18-hACE2 Mouse

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) not only affects the respiratory tract but also causes neurological symptoms such as loss of smell and taste, headache, fatigue or severe cerebrovascular complications. Using transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2), we investigated the spatiotemporal distribution and pathomorphological features in the CNS following intranasal infection with SARS-CoV-2 variants, as well as after prior influenza A virus infection. Apart from Omicron, we found all variants to frequently spread to and within the CNS. Infection was restricted to neurons and appeared to spread from the olfactory bulb mainly in basally oriented regions in the brain and into the spinal cord, independent of ACE2 expression and without evidence of neuronal cell death, axonal damage or demyelination. However, microglial activation, microgliosis and a mild macrophage and T cell dominated inflammatory response was consistently observed, accompanied by apoptotic death of endothelial, microglial and immune cells, without their apparent infection. Microgliosis and immune cell apoptosis indicate a potential role of microglia for pathogenesis and viral effect in COVID-19 and the possible impairment of neurological functions, especially in long COVID. These data may also be informative for the selection of therapeutic candidates and broadly support the investigation of agents with adequate penetration into relevant regions of the CNS.     

Chemiluminescence Immunoassay Based Serological Immunoassays for Detection of SARS-CoV-2 Neutralizing Antibodies in COVID-19 Convalescent Patients and Vaccinated Population

The development of rapid serological detection methods re urgently needed for determination of neutralizing antibodies in sera. In this study, four rapid methods (ACE2-RBD inhibition assay, S1-IgG detection, RBD-IgG detection, and N-IgG detection) were established and evaluated based on chemiluminescence technology. For the first time, a broadly neutralizing antibody with high affinity was used as a standard for the quantitative detection of SARS-CoV-2 specific neutralizing antibodies in human sera. Sera from COVID-19 convalescent patients (N = 119), vaccinated donors (N = 86), and healthy donors (N = 299) confirmed by microneutralization test (MNT) were used to evaluate the above methods. The result showed that the ACE2-RBD inhibition assay calculated with either ACE2-RBD binding inhibition percentage rate or ACE2-RBD inhibiting antibody concentration were strongly correlated with MNT (r ≥ 0.78, p < 0.0001) and also highly consistent with MNT (Kappa Value ≥ 0.94, p < 0.01). There was also a strong correlation between the two evaluation indices (r ≥ 0.99, p < 0.0001). Meanwhile, S1-IgG and RBD-IgG quantitative detection were also significantly correlated with MNT (r ≥ 0.73, p < 0.0001), and both methods were highly correlated with each other (r ≥ 0.95, p < 0.0001). However, the concentration of N-IgG antibodies showed a lower correlation with the MNT results (r < 0.49, p < 0.0001). The diagnostic assays presented here could be used for the evaluation of SARS-CoV-2 vaccine immunization effect and serological diagnosis of COVID-19 patients, and could also have guiding significance for establishing other rapid serological methods to surrogate neutralization tests for SARS-CoV-2.     

Factors Associated with Change in SARS-CoV-2 Antibody Titers from Three to Six Months after the Administration of the BNT162b2 mRNA COVID-19 Vaccine among Healthcare Workers in Japan: A Prospective Study

Objective We evaluated the change in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody titers from three to six months after the administration of the BNT162b2 vaccine among healthcare workers. Methods A total of 337 healthcare workers who received 2 doses of the BNT162b2 vaccine were included in this study. Factors associated with SARS-CoV-2 antibody titers at three and six months and the change in SARS-CoV-2 antibody titers between three and six months after vaccine administration were analyzed using a logistic regression analysis. Results The SARS-CoV-2 antibody titer at 3 months was 4,812.1±3,762.9 AU/mL in all subjects and was lower in older workers than in younger ones. The SARS-CoV-2 antibody titer at 6 months was 1,368.9±1,412.3 AU/mL in all subjects. The SARS-CoV-2 antibody titers that were found to be high at three months were also high at six months. The change in SARS-CoV-2 antibody titers from 3 to 6 months was -68.9%±16.1%. The higher SARS-CoV-2 antibody titers at three months showed a more marked decrease from three to six months than lower titers. Conclusion This study demonstrates that SARS-CoV-2 antibody titers at three months decreased with age and were associated with the antibody titers at six months and the change in titer from three to six months. Older individuals in particular need to be aware of the declining SARS-CoV-2 antibody titers at six months after the BNT162b2 vaccine. The results of this study may provide insight into COVID-19 vaccine booster strategies.     

Mucosal Vaccines,Sterilizing Immunity,and the Future of SARS-CoV-2 Virulence

Sterilizing immunity after vaccination is desirable to prevent the spread of infection from vaccinees, which can be especially dangerous in hospital settings while managing frail patients. Sterilizing immunity requires neutralizing antibodies at the site of infection, which for respiratory viruses such as SARS-CoV-2 implies the occurrence of neutralizing IgA in mucosal secretions. Systemic vaccination by intramuscular delivery induces no or low-titer neutralizing IgA against vaccine antigens. Mucosal priming or boosting, is needed to provide sterilizing immunity. On the other side of the coin, sterilizing immunity, by zeroing interhuman transmission, could confine SARS-CoV-2 in animal reservoirs, preventing spontaneous attenuation of virulence in humans as presumably happened with the endemic coronaviruses. We review here the pros and cons of each vaccination strategy, the current mucosal SARS-CoV-2 vaccines under development, and their implications for public health.     

Potential Autoimmunity Resulting from Molecular Mimicry between SARS-CoV-2 Spike and Human Proteins

Molecular mimicry between viral antigens and host proteins can produce cross-reacting antibodies leading to autoimmunity. The coronavirus SARS-CoV-2 causes COVID-19, a disease curiously resulting in varied symptoms and outcomes, ranging from asymptomatic to fatal. Autoimmunity due to cross-reacting antibodies resulting from molecular mimicry between viral antigens and host proteins may provide an explanation. Thus, we computationally investigated molecular mimicry between SARS-CoV-2 Spike and known epitopes. We discovered molecular mimicry hotspots in Spike and highlight two examples with tentative high autoimmune potential and implications for understanding COVID-19 complications. We show that a TQLPP motif in Spike and thrombopoietin shares similar antibody binding properties. Antibodies cross-reacting with thrombopoietin may induce thrombocytopenia, a condition observed in COVID-19 patients. Another motif, ELDKY, is shared in multiple human proteins, such as PRKG1 involved in platelet activation and calcium regulation, and tropomyosin, which is linked to cardiac disease. Antibodies cross-reacting with PRKG1 and tropomyosin may cause known COVID-19 complications such as blood-clotting disorders and cardiac disease, respectively. Our findings illuminate COVID-19 pathogenesis and highlight the importance of considering autoimmune potential when developing therapeutic interventions to reduce adverse reactions.     

COVID-19 vaccine effectiveness against SARS-CoV-2 infection during the Delta period,a nationwide study adjusting for chance of exposure,the Netherlands,July to December 2021

BackgroundDifferential SARS-CoV-2 exposure between vaccinated and unvaccinated individuals may confound vaccine effectiveness (VE) estimates.AimWe conducted a test-negative case–control study to determine VE against SARS-CoV-2 infection and the presence of confounding by SARS-CoV-2 exposure.MethodsWe included adults tested for SARS-CoV-2 at community facilities between 4 July and 8 December 2021 (circulation period of the Delta variant). The VE against SARS-CoV-2 infection after primary vaccination with an mRNA (Comirnaty or Spikevax) or vector-based vaccine (Vaxzevria or Janssen) was calculated using logistic regression adjusting for age, sex and calendar week (Model 1). We additionally adjusted for comorbidity and education level (Model 2) and SARS-CoV-2 exposure (number of close contacts, visiting busy locations, household size, face mask wearing, contact with SARS-CoV-2 case; Model 3). We stratified by age, vaccine type and time since vaccination.ResultsVE against infection (Model 3) was 64% (95% CI: 50–73), only slightly lower than in Models 1 (68%; 95% CI: 58–76) and 2 (67%; 95% CI: 56–75). Estimates stratified by age group, vaccine and time since vaccination remained similar: mRNA VE (Model 3) among people ≥ 50 years decreased significantly (p = 0.01) from 81% (95% CI: 66–91) at < 120 days to 61% (95% CI: 22–80) at ≥ 120 days after vaccination. It decreased from 83% to 59% in Model 1 and from 81% to 56% in Model 2.ConclusionSARS-CoV-2 exposure did not majorly confound the estimated COVID-19 VE against infection, suggesting that VE can be estimated accurately using routinely collected data without exposure information.     

Effectiveness of the Comirnaty (BNT162b2, BioNTech/Pfizer) vaccine in preventing SARS-CoV-2 infection among healthcare workers,Treviso province,Veneto region,Italy, 27 December 2020 to 24 March 2021

Data on effectiveness of the BioNTech­/Pfizer COVID-19 vaccine in real-world settings are limited. In a study of 6,423 healthcare workers in Treviso Province, Italy, we estimated that, within the time intervals of 14–21 days from the first and at least 7 days from the second dose, vaccine effectiveness in preventing SARS-CoV-2 infection was 84% (95% confidence interval (CI): 40–96) and 95% (95% CI: 62–99), respectively. These results could support the ongoing vaccination campaigns by providing evidence for targeted communication.