Structure-guided assembly of an influenza spike nanobicelle vaccine provides pan H1 intranasal protection.

Development of intranasal vaccines for respiratory viruses has gained popularity. However, currently only a live-attenuated influenza vaccine is FDA-approved for intranasal administration. Here, we focused on influenza virus as it circulates seasonally, has pandemic potential, and has vaccine formulations that present hemagglutinin (HA) in different structural arrangements. These display differences have not been correlated with induction of pan-H1 antibodies or shown to provide intranasal protection. Using electron microscopy, biochemistry and animal studies, we identified HA complexes arranged as lipid discs with multiple trimeric HAs displayed along the perimeter, termed spike nanobicelles (SNB). We utilized a structure-guided approach to synthesize in vitro assembled spiked nanobicelles (IA-SNB) from a classical 1934 H1N1 influenza virus. IA-SNBs elicited pan-H1 antibodies and provided protection against antigenically divergent H1N1 viruses via intranasal immunizations. Viral glycoprotein spikes displayed as SNBs could aid in combating antigenic variation and provide innovative intranasal vaccines to aid universal influenza vaccine development.

SARS-CoV-2 mucosal vaccine protects against clinical disease with sex bias in efficacy.

Intranasal mucosal vaccines can more effectively induce mucosal immune responses against SARS-CoV-2. Here, we show in hamsters that an intranasal subunit mucosal vaccine boost with the beta variant S1 can prevent weight loss, in addition to reducing viral load, which cannot be studied in macaques that don't develop COVID-like disease. Protective efficacy against both viral load and weight loss correlated with serum antibody titers. A sex bias was detected in that immune responses and protection against viral load were greater in females than males. We also found that priming with S1 from the Wuhan strain elicited lower humoral immune responses against beta variant and led to less protection against beta viral challenge, suggesting the importance of matched antigens. The greater efficacy of mucosal vaccines in the upper respiratory tract and the need to consider sex differences in vaccine protection are important in the development of future improved COVID-19 vaccines.

Durable immunity to SARS-CoV-2 in both lower and upper airways achieved with a gorilla adenovirus (GRAd) S-2P vaccine in non-human primates.

SARS-CoV-2 continues to pose a global threat, and current vaccines, while effective against severe illness, fall short in preventing transmission. To address this challenge, there's a need for vaccines that induce mucosal immunity and can rapidly control the virus. In this study, we demonstrate that a single immunization with a novel gorilla adenovirus-based vaccine (GRAd) carrying the pre-fusion stabilized Spike protein (S-2P) in non-human primates provided protective immunity for over one year against the BA.5 variant of SARS-CoV-2. A prime-boost regimen using GRAd followed by adjuvanted S-2P (GRAd+S-2P) accelerated viral clearance in both the lower and upper airways. GRAd delivered via aerosol (GRAd(AE)+S-2P) modestly improved protection compared to its matched intramuscular regimen, but showed dramatically superior boosting by mRNA and, importantly, total virus clearance in the upper airway by day 4 post infection. GrAd vaccination regimens elicited robust and durable systemic and mucosal antibody responses to multiple SARS-CoV-2 variants, but only GRAd(AE)+S-2P generated long-lasting T cell responses in the lung. This research underscores the flexibility of the GRAd vaccine platform to provide durable immunity against SARS-CoV-2 in both the lower and upper airways.

Corrigendum: Impact of adjuvant: trivalent vaccine with quadrivalent-like protection against heterologous Yamagata-lineage influenza B virus.

[This corrects the article DOI: 10.3389/fimmu.2022.1002286.].

Commercial influenza vaccines vary in HA-complex structure and in induction of cross-reactive HA antibodies.

Influenza virus infects millions of people annually and can cause global pandemics. Hemagglutinin (HA) is the primary component of commercial influenza vaccines (CIV), and antibody titer to HA is a primary correlate of protection. Continual antigenic variation of HA requires that CIVs are reformulated yearly. Structural organization of HA complexes have not previously been correlated with induction of broadly reactive antibodies, yet CIV formulations vary in how HA is organized. Using electron microscopy to study four current CIVs, we find structures including: individual HAs, starfish structures with up to 12 HA molecules, and novel spiked-nanodisc structures that display over 50 HA molecules along the complex's perimeter. CIV containing these spiked nanodiscs elicit the highest levels of heterosubtypic cross-reactive antibodies in female mice. Here, we report that HA structural organization can be an important CIV parameter and can be associated with the induction of cross-reactive antibodies to conserved HA epitopes.

Broadly neutralizing antibody induction by non-stabilized SARS-CoV-2 Spike mRNA vaccination in nonhuman primates.

Immunization with mRNA or viral vectors encoding spike with diproline substitutions (S-2P) has provided protective immunity against severe COVID-19 disease. How immunization with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) spike elicits neutralizing antibodies (nAbs) against difficult-to-neutralize variants of concern (VOCs) remains an area of great interest. Here, we compare immunization of macaques with mRNA vaccines expressing ancestral spike either including or lacking diproline substitutions, and show the diproline substitutions were not required for protection against SARS-CoV-2 challenge or induction of broadly neutralizing B cell lineages. One group of nAbs elicited by the ancestral spike lacking diproline substitutions targeted the outer face of the receptor binding domain (RBD), neutralized all tested SARS-CoV-2 VOCs including Omicron XBB.1.5, but lacked cross-Sarbecovirus neutralization. Structural analysis showed that the macaque broad SARS-CoV-2 VOC nAbs bound to the same epitope as a human broad SARS-CoV-2 VOC nAb, DH1193. Vaccine-induced antibodies that targeted the RBD inner face neutralized multiple Sarbecoviruses, protected mice from bat CoV RsSHC014 challenge, but lacked Omicron variant neutralization. Thus, ancestral SARS-CoV-2 spike lacking proline substitutions encoded by nucleoside-modified mRNA can induce B cell lineages binding to distinct RBD sites that either broadly neutralize animal and human Sarbecoviruses or recent Omicron VOCs.

Breadth of SARS-CoV-2 neutralization and protection induced by a nanoparticle vaccine.

Coronavirus vaccines that are highly effective against current and anticipated SARS-CoV-2 variants are needed to control COVID-19. We previously reported a receptor-binding domain (RBD)-sortase A-conjugated ferritin nanoparticle (scNP) vaccine that induced neutralizing antibodies against SARS-CoV-2 and pre-emergent sarbecoviruses and protected non-human primates (NHPs) from SARS-CoV-2 WA-1 infection. Here, we find the RBD-scNP induced neutralizing antibodies in NHPs against pseudoviruses of SARS-CoV and SARS-CoV-2 variants including 614G, Beta, Delta, Omicron BA.1, BA.2, BA.2.12.1, and BA.4/BA.5, and a designed variant with escape mutations, PMS20. Adjuvant studies demonstrate variant neutralization titers are highest with 3M-052-aqueous formulation (AF). Immunization twice with RBD-scNPs protect NHPs from SARS-CoV-2 WA-1, Beta, and Delta variant challenge, and protect mice from challenges of SARS-CoV-2 Beta variant and two other heterologous sarbecoviruses. These results demonstrate the ability of RBD-scNPs to induce broad neutralization of SARS-CoV-2 variants and to protect animals from multiple different SARS-related viruses. Such a vaccine could provide broad immunity to SARS-CoV-2 variants.

Impact of adjuvant: Trivalent vaccine with quadrivalent-like protection against heterologous Yamagata-lineage influenza B virus.

As new vaccine technologies and platforms, such as nanoparticles and novel adjuvants, are developed to aid in the establishment of a universal influenza vaccine, studying traditional influenza split/subunit vaccines should not be overlooked. Commercially available vaccines are typically studied in terms of influenza A H1 and H3 viruses but influenza B viruses need to be examined as well. Thus, there is a need to both understand the limitations of split/subunit vaccines and develop strategies to overcome those limitations, particularly their ability to elicit cross-reactive antibodies to the co-circulating Victoria (B-V) and Yamagata (B-Y) lineages of human influenza B viruses. In this study, we compared three commercial influenza hemagglutinin (HA) split/subunit vaccines, one quadrivalent (H1, H3, B-V, B-Y HAs) and two trivalent (H1, H3, B-V HAs), to characterize potential differences in their antibody responses and protection against a B-Y challenge. We found that the trivalent adjuvanted vaccine Fluad, formulated without B-Y HA, was able to produce antibodies to B-Y (cross-lineage) on a similar level to those elicited from a quadrivalent vaccine (Flucelvax) containing both B-V and B-Y HAs. Interestingly, Fluad protected mice from a lethal cross-lineage B-Y viral challenge, while another trivalent vaccine, Fluzone HD, failed to elicit antibodies or full protection following challenge. Fluad immunization also diminished viral burden in the lungs compared to Fluzone and saline groups. The success of a trivalent vaccine to provide protection from a cross-lineage influenza B challenge, similar to a quadrivalent vaccine, suggests that further analysis of different split/subunit vaccine formulations could identify mechanisms for vaccines to target antigenically different viruses. Understanding how to increase the breadth of the immune response following immunization will be needed for universal influenza vaccine development.

An intranasally administrated SARS-CoV-2 beta variant subunit booster vaccine prevents beta variant replication in rhesus macaques.

Emergence of SARS-CoV-2 variants and waning of vaccine/infection-induced immunity pose threats to curbing the COVID-19 pandemic. Effective, safe, and convenient booster vaccines are in need. We hypothesized that a variant-modified mucosal booster vaccine might induce local immunity to prevent SARS-CoV-2 infection at the port of entry. The beta-variant is one of the hardest to cross-neutralize. Herein, we assessed the protective efficacy of an intranasal booster composed of beta variant-spike protein S1 with IL-15 and TLR agonists in previously immunized macaques. The macaques were first vaccinated with Wuhan strain S1 with the same adjuvant. A total of 1 year later, negligibly detectable SARS-CoV-2-specific antibody remained. Nevertheless, the booster induced vigorous humoral immunity including serum- and bronchoalveolar lavage (BAL)-IgG, secretory nasal- and BAL-IgA, and neutralizing antibody against the original strain and/or beta variant. Beta-variant S1-specific CD4+ and CD8+ T cell responses were also elicited in PBMC and BAL. Following SARS-CoV-2 beta variant challenge, the vaccinated group demonstrated significant protection against viral replication in the upper and lower respiratory tracts, with almost full protection in the nasal cavity. The fact that one intranasal beta-variant booster administrated 1 year after the first vaccination provoked protective immunity against beta variant infections may inform future SARS-CoV-2 booster design and administration timing.

mRNA-1273 or mRNA-Omicron boost in vaccinated macaques elicits similar B cell expansion, neutralizing responses, and protection from Omicron.

SARS-CoV-2 Omicron is highly transmissible and has substantial resistance to neutralization following immunization with ancestral spike-matched vaccines. It is unclear whether boosting with Omicron-matched vaccines would enhance protection. Here, nonhuman primates that received mRNA-1273 at weeks 0 and 4 were boosted at week 41 with mRNA-1273 or mRNA-Omicron. Neutralizing titers against D614G were 4,760 and 270 reciprocal ID50 at week 6 (peak) and week 41 (preboost), respectively, and 320 and 110 for Omicron. 2 weeks after the boost, titers against D614G and Omicron increased to 5,360 and 2,980 for mRNA-1273 boost and 2,670 and 1,930 for mRNA-Omicron, respectively. Similar increases against BA.2 were observed. Following either boost, 70%-80% of spike-specific B cells were cross-reactive against WA1 and Omicron. Equivalent control of virus replication in lower airways was observed following Omicron challenge 1 month after either boost. These data show that mRNA-1273 and mRNA-Omicron elicit comparable immunity and protection shortly after the boost.

Breadth of SARS-CoV-2 Neutralization and Protection Induced by a Nanoparticle Vaccine.

Coronavirus vaccines that are highly effective against SARS-CoV-2 variants are needed to control the current pandemic. We previously reported a receptor-binding domain (RBD) sortase A-conjugated ferritin nanoparticle (RBD-scNP) vaccine that induced neutralizing antibodies against SARS-CoV-2 and pre-emergent sarbecoviruses and protected monkeys from SARS-CoV-2 WA-1 infection. Here, we demonstrate SARS-CoV-2 RBD-scNP immunization induces potent neutralizing antibodies in non-human primates (NHPs) against all eight SARS-CoV-2 variants tested including the Beta, Delta, and Omicron variants. The Omicron variant was neutralized by RBD-scNP-induced serum antibodies with a mean of 10.6-fold reduction of ID50 titers compared to SARS-CoV-2 D614G. Immunization with RBD-scNPs protected NHPs from SARS-CoV-2 WA-1, Beta, and Delta variant challenge, and protected mice from challenges of SARS-CoV-2 Beta variant and two other heterologous sarbecoviruses. These results demonstrate the ability of RBD-scNPs to induce broad neutralization of SARS-CoV-2 variants and to protect NHPs and mice from multiple different SARS-related viruses. Such a vaccine could provide the needed immunity to slow the spread of and reduce disease caused by SARS-CoV-2 variants such as Delta and Omicron.

Protection from SARS-CoV-2 Delta one year after mRNA-1273 vaccination in rhesus macaques coincides with anamnestic antibody response in the lung.

mRNA-1273 vaccine efficacy against SARS-CoV-2 Delta wanes over time; however, there are limited data on the impact of durability of immune responses on protection. Here, we immunized rhesus macaques and assessed immune responses over 1 year in blood and upper and lower airways. Serum neutralizing titers to Delta were 280 and 34 reciprocal ID50 at weeks 6 (peak) and 48 (challenge), respectively. Antibody-binding titers also decreased in bronchoalveolar lavage (BAL). Four days after Delta challenge, the virus was unculturable in BAL, and subgenomic RNA declined by ∼3-log10 compared with control animals. In nasal swabs, sgRNA was reduced by 1-log10, and the virus remained culturable. Anamnestic antibodies (590-fold increased titer) but not T cell responses were detected in BAL by day 4 post-challenge. mRNA-1273-mediated protection in the lungs is durable but delayed and potentially dependent on anamnestic antibody responses. Rapid and sustained protection in upper and lower airways may eventually require a boost.

Protection from SARS-CoV-2 Delta one year after mRNA-1273 vaccination in nonhuman primates is coincident with an anamnestic antibody response in the lower airway.

mRNA-1273 vaccine efficacy against SARS-CoV-2 Delta wanes over time; however, there are limited data on the impact of durability of immune responses on protection. We immunized rhesus macaques at weeks 0 and 4 and assessed immune responses over one year in blood, upper and lower airways. Serum neutralizing titers to Delta were 280 and 34 reciprocal ID 50 at weeks 6 (peak) and 48 (challenge), respectively. Antibody binding titers also decreased in bronchoalveolar lavage (BAL). Four days after challenge, virus was unculturable in BAL and subgenomic RNA declined ∼3-log 10 compared to control animals. In nasal swabs, sgRNA declined 1-log 10 and virus remained culturable. Anamnestic antibody responses (590-fold increase) but not T cell responses were detected in BAL by day 4 post-challenge. mRNA-1273-mediated protection in the lungs is durable but delayed and potentially dependent on anamnestic antibody responses. Rapid and sustained protection in upper and lower airways may eventually require a boost.

Protection against SARS-CoV-2 Beta variant in mRNA-1273 vaccine-boosted nonhuman primates.

Neutralizing antibody responses gradually wane against several variants of concern (VOCs) after vaccination with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine messenger RNA-1273 (mRNA-1273). We evaluated the immune responses in nonhuman primates that received a primary vaccination series of mRNA-1273 and were boosted about 6 months later with either homologous mRNA-1273 or heterologous mRNA-1273.ß, which encompasses the spike sequence of the B.1.351 Beta variant. After boost, animals had increased neutralizing antibody responses across all VOCs, which was sustained for at least 8 weeks after boost. Nine weeks after boost, animals were challenged with the SARS-CoV-2 Beta variant. Viral replication was low to undetectable in bronchoalveolar lavage and significantly reduced in nasal swabs in all boosted animals, suggesting that booster vaccinations may be required to sustain immunity and protection.

Chlamydia evasion of neutrophil host defense results in NLRP3 dependent myeloid-mediated sterile inflammation through the purinergic P2X7 receptor.

Chlamydia trachomatis infection causes severe inflammatory disease resulting in blindness and infertility. The pathophysiology of these diseases remains elusive but myeloid cell-associated inflammation has been implicated. Here we show NLRP3 inflammasome activation is essential for driving a macrophage-associated endometritis resulting in infertility by using a female mouse genital tract chlamydial infection model. We find the chlamydial parasitophorous vacuole protein CT135 triggers NLRP3 inflammasome activation via TLR2/MyD88 signaling as a pathogenic strategy to evade neutrophil host defense. Paradoxically, a consequence of CT135 mediated neutrophil killing results in a submucosal macrophage-associated endometritis driven by ATP/P2X7R induced NLRP3 inflammasome activation. Importantly, macrophage-associated immunopathology occurs independent of macrophage infection. We show chlamydial infection of neutrophils and epithelial cells produce elevated levels of extracellular ATP. We propose this source of ATP serves as a DAMP to activate submucosal macrophage NLRP3 inflammasome that drive damaging immunopathology. These findings offer a paradigm of sterile inflammation in infectious disease pathogenesis.

Immune correlates of protection by mRNA-1273 vaccine against SARS-CoV-2 in nonhuman primates.

Immune correlates of protection can be used as surrogate endpoints for vaccine efficacy. Here, nonhuman primates (NHPs) received either no vaccine or doses ranging from 0.3 to 100 µg of the mRNA-1273 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine. mRNA-1273 vaccination elicited circulating and mucosal antibody responses in a dose-dependent manner. Viral replication was significantly reduced in bronchoalveolar lavages and nasal swabs after SARS-CoV-2 challenge in vaccinated animals and most strongly correlated with levels of anti­S antibody and neutralizing activity. Lower antibody levels were needed for reduction of viral replication in the lower airway than in the upper airway. Passive transfer of mRNA-1273­induced immunoglobulin G to naïve hamsters was sufficient to mediate protection. Thus, mRNA-1273 vaccine­induced humoral immune responses are a mechanistic correlate of protection against SARS-CoV-2 in NHPs.

mRNA-1273 protects against SARS-CoV-2 beta infection in nonhuman primates.

B.1.351 is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant most resistant to antibody neutralization. We demonstrate how the dose and number of immunizations influence protection. Nonhuman primates received two doses of 30 or 100 µg of Moderna's mRNA-1273 vaccine, a single immunization of 30 µg, or no vaccine. Two doses of 100 µg of mRNA-1273 induced 50% inhibitory reciprocal serum dilution neutralizing antibody titers against live SARS-CoV-2 p.Asp614Gly and B.1.351 of 3,300 and 240, respectively. Higher neutralizing responses against B.1.617.2 were also observed after two doses compared to a single dose. After challenge with B.1.351, there was ~4- to 5-log10 reduction of viral subgenomic RNA and low to undetectable replication in bronchoalveolar lavages in the two-dose vaccine groups, with a 1-log10 reduction in nasal swabs in the 100-µg group. These data establish that a two-dose regimen of mRNA-1273 will be critical for providing upper and lower airway protection against major variants of concern.

Attenuated activation of pulmonary immune cells in mRNA-1273-vaccinated hamsters after SARS-CoV-2 infection.

The mRNA-1273 vaccine is effective against SARS-CoV-2 and was granted emergency use authorization by the FDA. Clinical studies, however, cannot provide the controlled response to infection and complex immunological insight that are only possible with preclinical studies. Hamsters are the only model that reliably exhibits severe SARS-CoV-2 disease similar to that in hospitalized patients, making them pertinent for vaccine evaluation. We demonstrate that prime or prime-boost administration of mRNA-1273 in hamsters elicited robust neutralizing antibodies, ameliorated weight loss, suppressed SARS-CoV-2 replication in the airways, and better protected against disease at the highest prime-boost dose. Unlike in mice and nonhuman primates, low-level virus replication in mRNA-1273-vaccinated hamsters coincided with an anamnestic response. Single-cell RNA sequencing of lung tissue permitted high-resolution analysis that is not possible in vaccinated humans. mRNA-1273 prevented inflammatory cell infiltration and the reduction of lymphocyte proportions, but enabled antiviral responses conducive to lung homeostasis. Surprisingly, infection triggered transcriptome programs in some types of immune cells from vaccinated hamsters that were shared, albeit attenuated, with mock-vaccinated hamsters. Our results support the use of mRNA-1273 in a 2-dose schedule and provide insight into the potential responses within the lungs of vaccinated humans who are exposed to SARS-CoV-2.

Protection against SARS-CoV-2 Beta Variant in mRNA-1273 Boosted Nonhuman Primates.

Neutralizing antibody responses gradually wane after vaccination with mRNA-1273 against several variants of concern (VOC), and additional boost vaccinations may be required to sustain immunity and protection. Here, we evaluated the immune responses in nonhuman primates that received 100 µg of mRNA-1273 vaccine at 0 and 4 weeks and were boosted at week 29 with mRNA-1273 (homologous) or mRNA-1273.ß (heterologous), which encompasses the spike sequence of the B.1.351 (beta or ß) variant. Reciprocal ID 50 pseudovirus neutralizing antibody geometric mean titers (GMT) against live SARS-CoV-2 D614G and the ß variant, were 4700 and 765, respectively, at week 6, the peak of primary response, and 644 and 553, respectively, at a 5-month post-vaccination memory time point. Two weeks following homologous or heterologous boost ß-specific reciprocal ID 50 GMT were 5000 and 3000, respectively. At week 38, animals were challenged in the upper and lower airway with the ß variant. Two days post-challenge, viral replication was low to undetectable in both BAL and nasal swabs in most of the boosted animals. These data show that boosting with the homologous mRNA-1273 vaccine six months after primary immunization provides up to a 20-fold increase in neutralizing antibody responses across all VOC, which may be required to sustain high-level protection against severe disease, especially for at-risk populations. ONE-SENTENCE SUMMARY: mRNA-1273 boosted nonhuman primates have increased immune responses and are protected against SARS-CoV-2 beta infection.

In vitro and in vivo functions of SARS-CoV-2 infection-enhancing and neutralizing antibodies.

SARS-CoV-2-neutralizing antibodies (NAbs) protect against COVID-19. A concern regarding SARS-CoV-2 antibodies is whether they mediate disease enhancement. Here, we isolated NAbs against the receptor-binding domain (RBD) or the N-terminal domain (NTD) of SARS-CoV-2 spike from individuals with acute or convalescent SARS-CoV-2 or a history of SARS-CoV infection. Cryo-electron microscopy of RBD and NTD antibodies demonstrated function-specific modes of binding. Select RBD NAbs also demonstrated Fc receptor-γ (FcγR)-mediated enhancement of virus infection in vitro, while five non-neutralizing NTD antibodies mediated FcγR-independent in vitro infection enhancement. However, both types of infection-enhancing antibodies protected from SARS-CoV-2 replication in monkeys and mice. Three of 46 monkeys infused with enhancing antibodies had higher lung inflammation scores compared to controls. One monkey had alveolar edema and elevated bronchoalveolar lavage inflammatory cytokines. Thus, while in vitro antibody-enhanced infection does not necessarily herald enhanced infection in vivo, increased lung inflammation can rarely occur in SARS-CoV-2 antibody-infused macaques.