Imprinting of serum neutralizing antibodies by Wuhan-1 mRNA vaccines.

Immune imprinting is a phenomenon in which prior antigenic experiences influence responses to subsequent infection or vaccination1,2. The effects of immune imprinting on serum antibody responses after boosting with variant-matched SARS-CoV-2 vaccines remain uncertain. Here, we characterized the serum antibody responses after mRNA vaccine boosting of mice and human clinical trial participants. In mice, a single dose of a preclinical version of mRNA-1273 vaccine encoding Wuhan-1 spike minimally imprinted serum responses elicited by Omicron boosters, enabling generation of type-specific antibodies. However, imprinting was observed in mice receiving an Omicron booster after two priming doses of mRNA-1273, an effect that was mitigated by a second booster dose of Omicron vaccine. In both SARS-CoV-2 infected or uninfected humans who received two Omicron-matched boosters after two or more doses of the prototype mRNA-1273 vaccine, spike-binding and neutralizing serum antibodies cross-reacted with Omicron variants as well as more distantly related sarbecoviruses. Because serum neutralizing responses against Omicron strains and other sarbecoviruses were abrogated after pre-clearing with Wuhan-1 spike protein, antibodies induced by XBB.1.5 boosting in humans focus on conserved epitopes targeted by the antecedent mRNA-1273 primary series. Thus, the antibody response to Omicron-based boosters in humans is imprinted by immunizations with historical mRNA-1273 vaccines, but this outcome may be beneficial as it drives expansion of cross-neutralizing antibodies that inhibit infection of emerging SARS-CoV-2 variants and distantly related sarbecoviruses.

Mucosal vaccine-induced cross-reactive CD8+ T cells protect against SARS-CoV-2 XBB.1.5 respiratory tract infection.

A nasally delivered chimpanzee adenoviral-vectored severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine (ChAd-SARS-CoV-2-S) is currently used in India (iNCOVACC). Here, we update this vaccine by creating ChAd-SARS-CoV-2-BA.5-S, which encodes a prefusion-stabilized BA.5 spike protein. Whereas serum neutralizing antibody responses induced by monovalent or bivalent adenoviral vaccines were poor against the antigenically distant XBB.1.5 strain and insufficient to protect in passive transfer experiments, mucosal antibody and cross-reactive memory T cell responses were robust, and protection was evident against WA1/2020 D614G and Omicron variants BQ.1.1 and XBB.1.5 in mice and hamsters. However, depletion of memory CD8+ T cells before XBB.1.5 challenge resulted in loss of protection against upper and lower respiratory tract infection. Thus, nasally delivered vaccines stimulate mucosal immunity against emerging SARS-CoV-2 strains, and cross-reactive memory CD8+ T cells mediate protection against lung infection by antigenically distant strains in the setting of low serum levels of cross-reactive neutralizing antibodies.

Intestinal helminth infection impairs vaccine-induced T cell responses and protection against SARS-CoV-2.

Although vaccines have reduced COVID-19 disease burden, their efficacy in helminth infection endemic areas is not well characterized. We evaluated the impact of infection by Heligmosomoides polygyrus bakeri (Hpb), a murine intestinal hookworm, on the efficacy of an mRNA vaccine targeting the Wuhan-1 spike protein of SARS-CoV-2. Although immunization generated similar B cell responses in Hpb-infected and uninfected mice, polyfunctional CD4+ and CD8+ T cell responses were markedly reduced in Hpb-infected mice. Hpb-infected and mRNA vaccinated mice were protected against the ancestral SARS-CoV-2 strain WA1/2020, but control of lung infection was diminished against an Omicron variant compared to animals immunized without Hpb infection. Helminth mediated suppression of spike-specific CD8+ T cell responses occurred independently of STAT6 signaling, whereas blockade of IL-10 rescued vaccine-induced CD8+ T cell responses. In mice, intestinal helminth infection impairs vaccine induced T cell responses via an IL-10 pathway and compromises protection against antigenically shifted SARS-CoV-2 variants.

Immunoglobulin replacement products protect against SARS-CoV-2 infection in vivo despite poor neutralizing activity.

Immunoglobulin (IG) replacement products are used routinely in patients with immune deficiency and other immune dysregulation disorders who have poor responses to vaccination and require passive immunity conferred by commercial antibody products. The binding, neutralizing, and protective activity of intravenously administered IG against SARS-CoV-2 emerging variants remains unknown. Here, we tested 198 different IG products manufactured from December 2019 to August 2022. We show that prepandemic IG had no appreciable cross-reactivity or neutralizing activity against SARS-CoV-2. Anti-spike antibody titers and neutralizing activity against SARS-CoV-2 WA1/2020 D614G increased gradually after the pandemic started and reached levels comparable to vaccinated healthy donors 18 months after the diagnosis of the first COVID-19 case in the United States in January 2020. The average time between production to infusion of IG products was 8 months, which resulted in poor neutralization of the variant strain circulating at the time of infusion. Despite limited neutralizing activity, IG prophylaxis with clinically relevant dosing protected susceptible K18-hACE2-transgenic mice against clinical disease, lung infection, and lung inflammation caused by the XBB.1.5 Omicron variant. Moreover, following IG prophylaxis, levels of XBB.1.5 infection in the lung were higher in FcγR-KO mice than in WT mice. Thus, IG replacement products with poor neutralizing activity against evolving SARS-CoV-2 variants likely confer protection to patients with immune deficiency disorders through Fc effector function mechanisms.

Avian influenza A viruses exhibit plasticity in sialylglycoconjugate receptor usage in human lung cells.

IMPORTANCE: It is well known that influenza A viruses (IAV) initiate host cell infection by binding to sialic acid, a sugar molecule present at the ends of various sugar chains called glycoconjugates. These sugar chains can vary in chain length, structure, and composition. However, it remains unknown if IAV strains preferentially bind to sialic acid on specific glycoconjugate type(s) for host cell infection. Here, we utilized CRISPR gene editing to abolish sialic acid on different glycoconjugate types in human lung cells, and evaluated human versus avian IAV infections. Our studies show that both human and avian IAV strains can infect human lung cells by utilizing any of the three major sialic acid-containing glycoconjugate types, specifically N-glycans, O-glycans, and glycolipids. Interestingly, simultaneous elimination of sialic acid on all three major glycoconjugate types in human lung cells dramatically decreased human IAV infection, yet had little effect on avian IAV infection. These studies show that avian IAV strains effectively utilize other less prevalent glycoconjugates for infection, whereas human IAV strains rely on a limited repertoire of glycoconjugate types. The remarkable ability of avian IAV strains to utilize diverse glycoconjugate types may allow for easy transmission into new host species.

A bivalent ChAd nasal vaccine protects against SARS-CoV-2 BQ.1.1 and XBB.1.5 infection and disease in mice and hamsters.

We previously described a nasally delivered monovalent adenoviral-vectored SARS-CoV-2 vaccine (ChAd-SARS-CoV-2-S, targeting Wuhan-1 spike [S]; iNCOVACC®) that is currently used in India as a primary or booster immunization. Here, we updated the mucosal vaccine for Omicron variants by creating ChAd-SARS-CoV-2-BA.5-S, which encodes for a pre-fusion and surface-stabilized S protein of the BA.5 strain, and then tested monovalent and bivalent vaccines for efficacy against circulating variants including BQ.1.1 and XBB.1.5. Whereas monovalent ChAd-vectored vaccines effectively induced systemic and mucosal antibody responses against matched strains, the bivalent ChAd-vectored vaccine elicited greater breadth. However, serum neutralizing antibody responses induced by both monovalent and bivalent vaccines were poor against the antigenically distant XBB.1.5 Omicron strain and did not protect in passive transfer experiments. Nonetheless, nasally delivered bivalent ChAd-vectored vaccines induced robust antibody and spike-specific memory T cell responses in the respiratory mucosa, and conferred protection against WA1/2020 D614G and Omicron variants BQ.1.1 and XBB.1.5 in the upper and lower respiratory tracts of both mice and hamsters. Our data suggest that a nasally delivered bivalent adenoviral-vectored vaccine induces protective mucosal and systemic immunity against historical and emerging SARS-CoV-2 strains without requiring high levels of serum neutralizing antibody.

Bivalent SARS-CoV-2 mRNA vaccines increase breadth of neutralization and protect against the BA.5 Omicron variant in mice.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants in the Omicron lineage has resulted in diminished Coronavirus Disease 2019 (COVID-19) vaccine efficacy and persistent transmission. In this study, we evaluated the immunogenicity and protective efficacy of two, recently authorized, bivalent COVID-19 vaccines that contain two mRNAs encoding Wuhan-1 and either BA.1 (mRNA-1273.214) or BA.4/5 (mRNA-1273.222) spike proteins. As a primary two-dose immunization series in mice, both bivalent vaccines induced greater neutralizing antibody responses against Omicron variants than the parental, monovalent mRNA-1273 vaccine. When administered to mice as a booster at 7 months after the primary vaccination series with mRNA-1273, the bivalent vaccines induced broadly neutralizing antibody responses. Whereas most anti-Omicron receptor binding domain antibodies in serum induced by mRNA-1273, mRNA-1273.214 and mRNA-1273.222 boosters cross-reacted with the antecedent Wuhan-1 spike antigen, the mRNA-1273.214 and mRNA-1273.222 bivalent vaccine boosters also induced unique BA.1-specific and BA.4/5-specific responses, respectively. Although boosting with parental or bivalent mRNA vaccines substantially improved protection against BA.5 compared to mice receiving two vaccine doses, the levels of infection, inflammation and pathology in the lung were lowest in animals administered the bivalent mRNA vaccines. Thus, boosting with bivalent Omicron-based mRNA-1273.214 or mRNA-1273.222 vaccines enhances immunogenicity and confers protection in mice against a currently circulating SARS-CoV-2 strain.

mRNA vaccine boosting enhances antibody responses against SARS-CoV-2 Omicron variant in individuals with antibody deficiency syndromes.

Individuals with primary antibody deficiency (PAD) syndromes have poor humoral immune responses requiring immunoglobulin replacement therapy. We followed individuals with PAD after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination by evaluating their immunoglobulin replacement products and serum for anti-spike binding, Fcγ receptor (FcγR) binding, and neutralizing activities. The immunoglobulin replacement products tested have low anti-spike and receptor-binding domain (RBD) titers and neutralizing activity. In coronavirus disease 2019 (COVID-19)-naive individuals with PAD, anti-spike and RBD titers increase after mRNA vaccination but wane by 90 days. Those vaccinated after SARS-CoV-2 infection develop higher and more sustained responses comparable with healthy donors. Most vaccinated individuals with PAD have serum-neutralizing antibody titers above an estimated correlate of protection against ancestral SARS-CoV-2 and Delta virus but not against Omicron virus, although this is improved by boosting. Thus, some immunoglobulin replacement products likely have limited protective activity, and immunization and boosting of individuals with PAD with mRNA vaccines should confer at least short-term immunity against SARS-CoV-2 variants, including Omicron.

mRNA-1273 and Ad26.COV2.S vaccines protect against the B.1.621 variant of SARS-CoV-2.

BACKGROUND: Since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019, viral variants with greater transmissibility or immune-evasion properties have arisen, which could jeopardize recently deployed vaccine- and antibody-based countermeasures. METHODS: Here, we evaluated in mice and hamsters the efficacy of a pre-clinical version of the Moderna mRNA vaccine (mRNA-1273) and the Johnson & Johnson recombinant adenoviral-vectored vaccine (Ad26.COV2.S) against the B.1.621 (Mu) variant of SARS-CoV-2, which contains spike mutations T95I, Y144S, Y145N, R346K, E484K, N501Y, D614G, P681H, and D950N. FINDINGS: Immunization of 129S2 and K18-human ACE2 transgenic mice with the mRNA-1273 vaccine protected against weight loss, lung infection, and lung pathology after challenge with the B.1.621 or WA1/2020 N501Y/D614G SARS-CoV-2 strain. Similarly, immunization of 129S2 mice and Syrian hamsters with a high dose of Ad26.COV2.S reduced lung infection after B.1.621 virus challenge. CONCLUSIONS: Thus, immunity induced by the mRNA-1273 or Ad26.COV2.S vaccine can protect against the B.1.621 variant of SARS-CoV-2 in multiple animal models. FUNDING: This study was supported by the NIH (R01 AI157155 and U01 AI151810), NIAID Centers of Excellence for Influenza Research and Response [CEIRR] contracts 75N93021C00014 and 75N93021C00016, and the Collaborative Influenza Vaccine Innovation Centers [CIVIC] contract 75N93019C00051. It was also supported, in part, by the National Institutes of Allergy and Infectious Diseases Center for Research on Influenza Pathogenesis (HHSN272201400008C) and the Japan Program for Infectious Diseases Research and Infrastructure (JP21wm0125002) from the Japan Agency for Medical Research and Development (AMED).

Boosting with variant-matched or historical mRNA vaccines protects against Omicron infection in mice.

The large number of spike substitutions in Omicron lineage variants (BA.1, BA.1.1., and BA.2) could jeopardize the efficacy of SARS-CoV-2 vaccines. We evaluated in mice the protective efficacy of the Moderna mRNA-1273 vaccine against BA.1 before or after boosting. Whereas two doses of mRNA-1273 vaccine induced high levels of neutralizing antibodies against historical WA1/2020 strains, lower levels against BA.1 were associated with breakthrough infection and inflammation in the lungs. A primary vaccination series with mRNA-1273.529, an Omicron-matched vaccine, potently neutralized BA.1 but inhibited historical or other SARS-CoV-2 variants less effectively. However, boosting with either mRNA-1273 or mRNA-1273.529 vaccines increased neutralizing titers and protection against BA.1 and BA.2 infection. Nonetheless, the neutralizing antibody titers were higher, and lung viral burden and cytokines were slightly lower in mice boosted with mRNA-1273.529 and challenged with BA.1. Thus, boosting with mRNA-1273 or mRNA-1273.529 enhances protection against Omicron infection with limited differences in efficacy measured.

Boosting with Omicron-matched or historical mRNA vaccines increases neutralizing antibody responses and protection against B.1.1.529 infection in mice.

The B.1.1.529 Omicron variant jeopardizes vaccines designed with early pandemic spike antigens. Here, we evaluated in mice the protective activity of the Moderna mRNA-1273 vaccine against B.1.1.529 before or after boosting with preclinical mRNA-1273 or mRNA-1273.529, an Omicron-matched vaccine. Whereas two doses of mRNA-1273 vaccine induced high levels of serum neutralizing antibodies against historical WA1/2020 strains, levels were lower against B.1.1.529 and associated with infection and inflammation in the lung. A primary vaccination series with mRNA-1273.529 potently neutralized B.1.1.529 but showed limited inhibition of historical or other SARS-CoV-2 variants. However, boosting with mRNA-1273 or mRNA-1273.529 vaccines increased serum neutralizing titers and protection against B.1.1.529 infection. Nonetheless, the levels of inhibitory antibodies were higher, and viral burden and cytokines in the lung were slightly lower in mice given the Omicron-matched mRNA booster. Thus, in mice, boosting with mRNA-1273 or mRNA-1273.529 enhances protection against B.1.1.529 infection with limited differences in efficacy measured.

Protective activity of mRNA vaccines against ancestral and variant SARS-CoV-2 strains.

Although mRNA vaccines encoding the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) prevent COVID-19, the emergence of new viral variants jeopardizes their efficacy. Here, we assessed the immunogenicity and protective activity of historical (mRNA-1273, designed for Wuhan-1 spike protein) or modified (mRNA-1273.351, designed for B.1.351 spike protein) Moderna mRNA vaccines in 129S2 and K18-hACE2 mice. Mice were immunized with either high-dose or low-dose formulations of the mRNA vaccines, where low-dose vaccination modeled suboptimal immune responses. Immunization with formulations at either dose induced neutralizing antibodies in serum against ancestral SARS-CoV-2 WA1/2020 and several virus variants, although serum titers were lower against the B.1.617.2 (Delta) virus. Protection against weight loss and lung pathology was observed with all high-dose vaccines against all viruses. However, low-dose formulations of the vaccines, which produced lower magnitude antibody and T cell responses, showed breakthrough lung infections with B.1.617.2 and development of pneumonia in K18-hACE2 mice. Thus, in individuals with reduced immunity after mRNA vaccination, breakthrough infection and disease may occur with some SARS-CoV-2 variants.

SARS-CoV-2 booster vaccination rescues attenuated IgG1 memory B cell response in primary antibody deficiency patients.

Background: Although SARS-CoV-2 vaccines have proven effective in eliciting a protective immune response in healthy individuals, their ability to induce a durable immune response in immunocompromised individuals remains poorly understood. Primary antibody deficiency (PAD) syndromes are among the most common primary immunodeficiency disorders in adults and are characterized by hypogammaglobulinemia and impaired ability to mount robust antibody responses following infection or vaccination. Methods: Here, we present an analysis of both the B and T cell response in a prospective cohort of 30 individuals with PAD up to 150 days following initial COVID-19 vaccination and 150 days post mRNA booster vaccination. Results: After the primary vaccination series, many of the individuals with PAD syndromes mounted SARS-CoV-2 specific memory B and CD4+ T cell responses that overall were comparable to healthy individuals. Nonetheless, individuals with PAD syndromes had reduced IgG1+ and CD11c+ memory B cell responses following the primary vaccination series, with the defect in IgG1 class-switching rescued following mRNA booster doses. Boosting also elicited an increase in the SARS-CoV-2-specific B and T cell response and the development of Omicron-specific memory B cells in COVID-19-naïve PAD patients. Individuals that lacked detectable B cell responses following primary vaccination did not benefit from booster vaccination. Conclusion: Together, these data indicate that SARS-CoV-2 vaccines elicit memory B and T cells in most PAD patients and highlights the importance of booster vaccination in immunodeficient individuals.

Protective activity of mRNA vaccines against ancestral and variant SARS-CoV-2 strains.

Although mRNA vaccines prevent COVID-19, variants jeopardize their efficacy as immunity wanes. Here, we assessed the immunogenicity and protective activity of historical (mRNA-1273, designed for Wuhan-1 spike) or modified (mRNA-1273.351, designed for B.1.351 spike) preclinical Moderna mRNA vaccines in 129S2 and K18-hACE2 mice. Immunization with high or low dose formulations of mRNA vaccines induced neutralizing antibodies in serum against ancestral SARS-CoV-2 and several variants, although levels were lower particularly against the B.1.617.2 (Delta) virus. Protection against weight loss and lung pathology was observed with all high-dose vaccines against all viruses. Nonetheless, low-dose formulations of the vaccines, which produced lower magnitude antibody and T cell responses, and serve as a possible model for waning immunity, showed breakthrough lung infection and pneumonia with B.1.617.2. Thus, as levels of immunity induced by mRNA vaccines decline, breakthrough infection and disease likely will occur with some SARS-CoV-2 variants, suggesting a need for additional booster regimens.

Host factor Rab11a is critical for efficient assembly of influenza A virus genomic segments.

It is well documented that influenza A viruses selectively package 8 distinct viral ribonucleoprotein complexes (vRNPs) into each virion; however, the role of host factors in genome assembly is not completely understood. To evaluate the significance of cellular factors in genome assembly, we generated a reporter virus carrying a tetracysteine tag in the NP gene (NP-Tc virus) and assessed the dynamics of vRNP localization with cellular components by fluorescence microscopy. At early time points, vRNP complexes were preferentially exported to the MTOC; subsequently, vRNPs associated on vesicles positive for cellular factor Rab11a and formed distinct vRNP bundles that trafficked to the plasma membrane on microtubule networks. In Rab11a deficient cells, however, vRNP bundles were smaller in the cytoplasm with less co-localization between different vRNP segments. Furthermore, Rab11a deficiency increased the production of non-infectious particles with higher RNA copy number to PFU ratios, indicative of defects in specific genome assembly. These results indicate that Rab11a+ vesicles serve as hubs for the congregation of vRNP complexes and enable specific genome assembly through vRNP:vRNP interactions, revealing the importance of Rab11a as a critical host factor for influenza A virus genome assembly.

Robust induction of TRMs by combinatorial nanoshells confers cross-strain sterilizing immunity against lethal influenza viruses.

Antigen-specific lung-resident memory T cells (TRMs) constitute the first line of defense that mediates rapid protection against respiratory pathogens and inspires novel vaccine designs against infectious pandemic threats, yet effective means of inducing TRMs, particularly via non-viral vectors, remain challenging. Here, we demonstrate safe and potent induction of lung-resident TRMs using a biodegradable polymeric nanoshell that co-encapsulates antigenic peptides and TLR9 agonist CpG-oligodeoxynucleotide (CpG-ODN) in a virus-mimicking structure. Through subcutaneous priming and intranasal boosting, the combinatorial nanoshell vaccine elicits prominent lung-resident CD4+ and CD8+ T cells that surprisingly show better durability than live viral infections. In particular, nanoshells containing CpG-ODN and a pair of conserved class I and II major histocompatibility complex-restricted influenza nucleoprotein-derived antigenic peptides are demonstrated to induce near-sterilizing immunity against lethal infections with influenza A viruses of different strains and subtypes in mice, resulting in rapid elimination of replicating viruses. We further examine the pulmonary transport dynamic and optimal composition of the nanoshell vaccine conducive for robust TRM induction as well as the benefit of subcutaneous priming on TRM replenishment. The study presents a practical vaccination strategy for inducing protective TRM-mediated immunity, offering a compelling platform and critical insights in the ongoing quest toward a broadly protective vaccine against universal influenza as well as other respiratory pathogens.

The p150 Isoform of ADAR1 Blocks Sustained RLR signaling and Apoptosis during Influenza Virus Infection.

Signaling through retinoic acid inducible gene I (RIG-I) like receptors (RLRs) is tightly regulated, with activation occurring upon sensing of viral nucleic acids, and suppression mediated by negative regulators. Under homeostatic conditions aberrant activation of melanoma differentiation-associated protein-5 (MDA5) is prevented through editing of endogenous dsRNA by RNA editing enzyme Adenosine Deaminase Acting on RNA (ADAR1). In addition, ADAR1 is postulated to play pro-viral and antiviral roles during viral infections that are dependent or independent of RNA editing activity. Here, we investigated the importance of ADAR1 isoforms in modulating influenza A virus (IAV) replication and revealed the opposing roles for ADAR1 isoforms, with the nuclear p110 isoform restricting versus the cytoplasmic p150 isoform promoting IAV replication. Importantly, we demonstrate that p150 is critical for preventing sustained RIG-I signaling, as p150 deficient cells showed increased IFN-ß expression and apoptosis during IAV infection, independent of RNA editing activity. Taken together, the p150 isoform of ADAR1 is important for preventing sustained RIG-I induced IFN-ß expression and apoptosis during viral infection.

Vaccine-induced antigen-specific regulatory T cells attenuate the antiviral immunity against acute influenza virus infection.

Peptide-based T cell vaccines targeting the conserved epitopes of influenza virus can provide cross-protection against distantly related strains, but they are generally not immunogenic. Foreign antigen-specific regulatory T (Treg) cells are induced under subimmunogenic conditions peripherally, although their development and role in vaccine-mediated antiviral immunity is unclear. Here, we demonstrated primary vaccination with peptides alone significantly induced antigen-specific Foxp3+ Treg cells, which were further expanded by repeated vaccination with unadjuvanted peptides. Certain adjuvants, including CpG, suppressed the induction and expansion of antigen-specific Treg cells by peptide vaccination. Interestingly, secondary influenza virus infection significantly increased the frequency of preexisting antigen-specific Treg cells, although primary infection barely induced them. Importantly, specific depletion of vaccine-induced antigen-specific Treg cells promoted influenza viral clearance, indicating their inhibitory role in vivo. Immunization with CpG-adjuvanted peptides by the subcutaneous prime-intranasal-boost strategy restricted the recruitment and accumulation of antigen-specific Treg cells in lung, and stimulated robust T cell immunity. Finally, subcutaneous prime-intranasal-boost immunization with CpG-adjuvanted peptides or whole-inactivated influenza vaccines protected mice from heterosubtypic influenza virus infection. In conclusion, antigen-specific Treg cells induced by peptide vaccines attenuate the antiviral immunity against influenza virus infection. CpG-adjuvanted peptide vaccines provide heterosubtypic influenza protection probably by inhibiting Treg development and enhancing T cell immunity.