The role of vaccination route with an adenovirus-vectored vaccine in protection, viral control, and transmission in the SARS-CoV-2/K18-hACE2 mouse infection model.

Introduction: Vaccination is the most effective mechanism to prevent severe COVID-19. However, breakthrough infections and subsequent transmission of SARS-CoV-2 remain a significant problem. Intranasal vaccination has the potential to be more effective in preventing disease and limiting transmission between individuals as it induces potent responses at mucosal sites. Methods: Utilizing a replication-deficient adenovirus serotype 5-vectored vaccine expressing the SARS-CoV-2 RBD (AdCOVID) in homozygous and heterozygous transgenic K18-hACE2, we investigated the impact of the route of administration on vaccine immunogenicity, SARS-CoV-2 transmission, and survival. Results: Mice vaccinated with AdCOVID via the intramuscular or intranasal route and subsequently challenged with SARS-CoV-2 showed that animals vaccinated intranasally had improved cellular and mucosal antibody responses. Additionally, intranasally vaccinated animals had significantly better viremic control, and protection from lethal infection compared to intramuscularly vaccinated animals. Notably, in a novel transmission model, intranasal vaccination reduced viral transmission to naïve co-housed mice compared to intramuscular vaccination. Discussion: Our data provide convincing evidence for the use of intranasal vaccination in protecting against SARS-CoV-2 infection and transmission.

A single intranasal administration of AdCOVID protects against SARS-CoV-2 infection in the upper and lower respiratory tracts.

The coronavirus disease 2019 (COVID-19) pandemic has illustrated the critical need for effective prophylactic vaccination to prevent the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Intranasal vaccination is an attractive approach for preventing COVID-19 as the nasal mucosa is the site of initial SARS-CoV-2 entry and viral replication prior to aspiration into the lungs. We previously demonstrated that a single intranasal administration of a candidate adenovirus type 5-vectored vaccine encoding the receptor-binding domain of the SARS-CoV-2 spike protein (AdCOVID) induced robust immunity in both the airway mucosa and periphery, and completely protected K18-hACE2 mice from lethal SARS-CoV-2 challenge. Here we show that a single intranasal administration of AdCOVID limits viral replication in the nasal cavity of K18-hACE2 mice. AdCOVID also induces sterilizing immunity in the lungs of mice as reflected by the absence of infectious virus. Finally, AdCOVID prevents SARS-CoV-2 induced pathological damage in the lungs of mice. These data show that AdCOVID not only limits viral replication in the respiratory tract, but it also prevents virus-induced inflammation and immunopathology following SARS-CoV-2 infection.

Single-Dose Intranasal Administration of AdCOVID Elicits Systemic and Mucosal Immunity against SARS-CoV-2 and Fully Protects Mice from Lethal Challenge.

The coronavirus disease 2019 (COVID-19) pandemic has highlighted the urgent need for effective prophylactic vaccination to prevent the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Intranasal vaccination is an attractive strategy to prevent COVID-19 as the nasal mucosa represents the first-line barrier to SARS-CoV-2 entry. The current intramuscular vaccines elicit systemic immunity but not necessarily high-level mucosal immunity. Here, we tested a single intranasal dose of our candidate adenovirus type 5-vectored vaccine encoding the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (AdCOVID) in inbred, outbred, and transgenic mice. A single intranasal vaccination with AdCOVID elicited a strong and focused immune response against RBD through the induction of mucosal IgA in the respiratory tract, serum neutralizing antibodies, and CD4+ and CD8+ T cells with a Th1-like cytokine expression profile. A single AdCOVID dose resulted in immunity that was sustained for over six months. Moreover, a single intranasal dose completely protected K18-hACE2 mice from lethal SARS-CoV-2 challenge, preventing weight loss and mortality. These data show that AdCOVID promotes concomitant systemic and mucosal immunity and represents a promising vaccine candidate.

Safety and Immunogenicity of a Novel Intranasal Influenza Vaccine (NasoVAX): A Phase 2 Randomized, Controlled Trial.

Annual influenza vaccination greatly reduces morbidity and mortality, but effectiveness remains sub-optimal. Weaknesses of current vaccines include low effectiveness against mismatched strains, lack of mucosal and other effective tissue-resident immune responses, weak cellular immune responses, and insufficiently durable immune responses. The safety and immunogenicity of NasoVAX, a monovalent intranasal influenza vaccine based on a replication-deficient adenovirus type 5 platform, were evaluated in a placebo-controlled single ascending-dose study. Sixty healthy adults (18-49 years) received a single intranasal dose of 1×109 viral particles (vp), 1 × 1010 vp, or 1 × 1011 vp of NasoVAX or placebo. NasoVAX was well-tolerated and elicited robust influenza-specific systemic and mucosal immune responses. The highest NasoVAX dose and the approved Fluzone® influenza vaccine elicited comparable hemagglutination inhibition (HAI) geometric mean titers (152.8 vs. 293.4) and microneutralization (MN) geometric mean titers (142.5 vs. 162.8), with NasoVAX HAI titers maintained more than 1-year on average following a single dose. Hemagglutinin-specific T cells responses were also documented in peripheral mononuclear cell (PBMC) preparations. Consistent with the intranasal route of administration, NasoVAX elicited antigen-specific mucosal IgA responses in the nasopharyngeal cavity with an increase of approximately 2-fold over baseline GMT at the mid- and high-doses. In summary, NasoVAX appeared safe and elicited a broad immune response, including humoral, cellular, and mucosal immunity, with no impact of baseline anti-adenovirus antibody at the most immunogenic dose.

Single-dose intranasal administration of AdCOVID elicits systemic and mucosal immunity against SARS-CoV-2 in mice.

The coronavirus disease 2019 (COVID-19) pandemic has highlighted the urgent need for effective preventive vaccination to reduce burden and spread of severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) in humans. Intranasal vaccination is an attractive strategy to prevent COVID-19 as the nasal mucosa represents the first-line barrier to SARS-CoV-2 entry before viral spread to the lung. Although SARS-CoV-2 vaccine development is rapidly progressing, the current intramuscular vaccines are designed to elicit systemic immunity without conferring mucosal immunity. Here, we show that AdCOVID, an intranasal adenovirus type 5 (Ad5)-vectored vaccine encoding the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, elicits a strong and focused immune response against RBD through the induction of mucosal IgA, serum neutralizing antibodies and CD4+ and CD8+ T cells with a Th1-like cytokine expression profile. Therefore, AdCOVID, which promotes concomitant systemic and local mucosal immunity, represents a promising COVID-19 vaccine candidate.

Nasal IgA Provides Protection against Human Influenza Challenge in Volunteers with Low Serum Influenza Antibody Titre.

In spite of there being a number of vaccines, influenza remains a significant global cause of morbidity and mortality. Understanding more about natural and vaccine induced immune protection against influenza infection would help to develop better vaccines. Virus specific IgG is a known correlate of protection, but other factors may help to reduce viral load or disease severity, for example IgA. In the current study we measured influenza specific responses in a controlled human infection model using influenza A/California/2009 (H1N1) as the challenge agent. Volunteers were pre-selected with low haemagglutination inhibition (HAI) titres in order to ensure a higher proportion of infection; this allowed us to explore the role of other immune correlates. In spite of HAI being uniformly low, there were variable levels of H1N1 specific IgG and IgA prior to infection. There was also a range of disease severity in volunteers allowing us to compare whether differences in systemic and local H1N1 specific IgG and IgA prior to infection affected disease outcome. H1N1 specific IgG level before challenge did not correlate with protection, probably due to the pre-screening for individuals with low HAI. However, the length of time infectious virus was recovered from the nose was reduced in patients with higher pre-existing H1N1 influenza specific nasal IgA or serum IgA. Therefore, IgA contributes to protection against influenza and should be targeted in vaccines.

Characterisation of a wild-type influenza (A/H1N1) virus strain as an experimental challenge agent in humans.

BACKGROUND: Human challenge models using respiratory viruses such as influenza are increasingly utilised in the development of novel vaccines and anti-viral modalities and can provide preliminary evidence of protection before evaluation in field trials. We describe the results of a clinical study characterising an A/H1N1 influenza challenge virus in humans. METHODS: The challenge agent, influenza A/California/2009 (H1N1), was manufactured under cGMP conditions and characterised in accordance with regulatory guidelines. A dose-ascending open-label clinical study was conducted in 29 healthy young adults screened sero-negative to the challenge strain. Subjects were intranasally inoculated with three increasing doses of virus and physician-reported signs, subjected-reported symptoms, viral shedding and immunological responses were monitored. RESULTS: A dose-dependent increase in clinical signs and symptoms was observed with 75% of subjects developing laboratory-confirmed illness at the highest inoculum (3.5 × 10(6) TCID50). At the highest dose, physician or subject-reported signs of infection were classified as mild (all subjects), moderate (50%) and severe (16%) with peak symptoms recorded four days after infection. Clinical signs were correlated with nasal mucus weight (P < .001) and subject-reported symptoms (P < .001). Geometric mean peak viral shedding was log10 5.16 TCID50 and occurred three days after inoculation with a median duration of five days. The safety profile was such that physiological responses to viral infection were mainly restricted to the upper airways but were not of such severity to be of clinical concern. CONCLUSIONS: A highly characterised wild-type Influenza A/California/2009 (H1N1) virus manufactured for clinical use was shown to induce a good infectivity profile in human volunteers. This clinical challenge model can be used for evaluating potential efficacy of vaccines and anti-viral therapeutics. TRIAL REGISTRATION: NCT02014870.

Increasing cellular immunogenicity to peptide-based vaccine candidates using a fluorocarbon antigen delivery system.

Traditionally, synthetic peptide vaccines for infectious diseases and cancer require adjuvants to achieve optimal immunogenicity. Here we describe a novel method of peptide modification using a fluorocarbon chain which can substantially increase peptide-specific cellular immune responses in the absence of adjuvant. We demonstrate that fluorocarbon-modified peptides (fluoropeptides) derived from HIV, influenza and hepatitis C virus can significantly increase interferon gamma ELISpot responses against cytotoxic and T-helper epitopes compared to unmodified peptides or lipopeptides in mice. Increases in both T-helper1 and T-helper2 cytokines are observed. Fluoropeptides show enhanced ability of the antigen to persist at the site of administration and persistence is associated with a prolonged and elevated immune response. Additionally we demonstrate that fluoropeptides have increased proteolytic resistance thereby potentially supporting their increased half-life in vivo. Fluorocarbon-modification of peptides provides a valuable tool for increasing cellular immunogenicity of vaccines for infectious diseases and cancer without requirement for traditional adjuvants.

A novel peptide-based pan-influenza A vaccine: a double blind, randomised clinical trial of immunogenicity and safety.

BACKGROUND: FP-01.1 is a novel synthetic influenza A vaccine consisting of six fluorocarbon-modified 35-mer peptides that encapsulate multiple CD4+ and CD8+ T-cell epitopes and is designed to induce an immune response across a broad population. METHODS: FP-01.1 was evaluated for safety and immunogenicity in a randomised, double-blind, placebo-controlled, dose-escalation, phase I clinical study in healthy adult volunteers (n=49). IFNγ ELISpot assays and multicolour flow cytometry were used to characterise the immune response. RESULTS: FP-01.1 was safe and well tolerated at all doses tested with a similar adverse event profile in actively vaccinated subjects compared with controls. Maximum immunogenicity was in the 150 µg/peptide dose group where a robust response (243 spots/million PBMC) was demonstrated in 75% subjects compared with 0% in placebo controls. All six peptides were immunogenic. FP-01.1 induced dual CD4+ and CD8+ T cell responses and vaccine-specific T cells cross-recognise divergent influenza strains. CONCLUSIONS: This first-in-human study showed that FP-01.1 has an acceptable safety and tolerability profile and generated robust anti-viral T cell responses in a high proportion of subjects tested. The results support the further clinical testing of FP-01.1 prior to clinical, proof-of-concept, live viral challenge studies.

Differential capacity of T cell priming in naive donors of promiscuous CD4+ T cell epitopes of HCV NS3 and Core proteins.

To understand the inter-individual and virus-independent variability of CD4+ T cell responses to HCV components, we evaluated the effect on these responses of HLA II molecules in uninfected healthy donors. Using HLA II-specific binding assays, we identified, in the Core and NS3 proteins, 21 long fragments and 24 15-mer peptides that bound to four to eight of the most preponderant HLA II molecules. We then evaluated the priming capacity of eight long promiscuous peptides in 12 HLA-unrelated healthy donors. The NS3 1250-1264 peptide primed T cells in all the naive donors, while five others were stimulating in at least half of the individuals. We also report sequences that bind to multiple HLA II molecules but are weakly immunogenic. We therefore conclude that (i) broad HLA II specificity is only a prerequisite for a peptide to be stimulating in multiple individuals, and (ii) promiscuous peptides widely differ in their capacity to prime CD4+ T cells from uninfected healthy donors. We suggest that these priming differences result from inter-individual variations in the peptide-specific T cell repertoire. Interestingly, five of the most immunogenic peptides we identified correspond to frequently targeted T cell epitopes in infected patients.

Determination of a HLA II promiscuous peptide cocktail as potential vaccine against EBV latency II malignancies.

The Epstein-Barr virus (EBV) is associated with several malignant diseases, which can be distinguished by their patterns of viral latent gene expression. The latency II program is limited to the expression of the nonimmunodominant antigens EBNA1, LMP1 and LMP2 and is seen in EBV-positive Hodgkin disease, nasopharyngeal carcinomas, and peripheral T/NK-cell lymphomas. CD4 T cells may play a crucial role in controlling these EBV latency II malignancies. In this study, we used the prediction software TEPITOPE to predict promiscuous major histocompatibility complex class II epitopes derived from the latency II antigens EBNA1, LMP1, and LMP2. The predicted peptides were then submitted to peptide-binding assays on HLA II purified molecules, which allowed the selection of 6 peptides (EBNA1: 3; LMP1: 1; and LMP2: 2) with a highly promiscuous capability of binding. This peptide cocktail was immunogenic in a model of HLA-DR1 transgenic mice, leading to a specific cellular and humoral TH1 response. The peptides were also recognized by human CD4 T cells from individuals expressing various HLA II genotypes. This promiscuous peptide cocktail could be immunogenic in the majority of the population and may be used as a peptide-based vaccine in EBV latency II malignancies.

Peptide-binding assays and HLA II transgenic Abeta degrees mice are consistent and complementary tools for identifying HLA II-restricted peptides.

The identification of MHC class II-restricted peptides has become a priority for the development of peptide-based prophylactic and therapeutic vaccines. The aim of this study was to assess the correlations between peptide-binding assays on purified HLA II molecules and immunization of human HLA II transgenic mice deficient in murine class II molecules (Abeta degrees ). We used as models two MHC class II-restricted peptides, one derived from the HIV Nef regulatory protein (Nef (56-68)) and the other from the Schistosoma mansoni 28-kDa glutathione-S-transferase (Sm28GST (190-211)). High correlations were found between the two approaches, which showed that the Nef (56-68) and Sm28GST (190-211) peptides may represent promiscuous ligands for HLA-DQ and for HLA-DR molecules, respectively. We suggest a rational method based on the combination of peptide-binding assays and HLA II transgenic mice experiments as consistent and complementary tools for selecting T helper epitopes.

HLA-DP4, the most frequent HLA II molecule, defines a new supertype of peptide-binding specificity.

Among HLA-DP specificities, HLA-DP4 specificity involves at least two molecules, HLA-DPA1*0103/DPB1*0401 (DP401) and HLA-DPA1*0103/DPB1*0402 (DP402), which differ from each other by only three residues. Together, they are present worldwide at an allelic frequency of 20-60% and are the most abundant human HLA II alleles. Strikingly, the peptide-binding specificities of these molecules have never been investigated. Hence, in this study, we report the peptide-binding motifs of both molecules. We first set up a binding assay specific for the immunopurified HLA-DP4 molecules. Using multiple sets of synthetic peptides, we successfully defined the amino acid preferences of the anchor residues. With these assays, we were also able to identify new peptide ligands from allergens and viral and tumor Ags. DP401 and DP402 exhibit very similar patterns of recognition in agreement with molecular modeling of the complexes. Pockets P1 and P6 accommodate the main anchor residues and interestingly contain only two polymorphic residues, beta86 and beta11, respectively. Both positions are almost dimorphic and thus produce a limited number of pocket combinations. Taken together, our results support the existence of three main binding supertypes among HLA-DP molecules and should significantly contribute to the identification of universal epitopes to be used in peptide-based vaccines for cancer, as well as for allergic or infectious diseases.

Evidences of conformational changes in class II Major Histocompatibility Complex molecules that affect the immunogenicity.

The N-terminal part of class II-associated invariant chain peptide (CLIP) is assumed to interact with an accessory peptide-binding site on the class II Major Histocompatibility Complex (MHC) molecule, and promote a conformational modification. We have linked this immunoregulatory segment (residues 81-88) to the N-terminus of the influenza hemagglutinin (HA) 307-319 epitope in order to evaluate relationships between the MHC conformational changes and their implication in immune responses. Our chimeric peptide, named CLIP-HA, bind with the same affinity to class II HLA-DR1 molecules as the HA peptide, and is normally recognized by HA-specific T cells. Interestingly, the presence of the N-terminal CLIP region enhances the rate of association to soluble DR1 molecules but prevents the formation of SDS-resistant complexes. These features suggest the existence of HLA-DR1 conformational changes induced by the chimeric peptide. Furthermore, while in vitro HA and CLIP-HA peptides associated to DR1 could not be differentiated based on T-cell recognition, in vivo the CLIP residues strongly impaired the immunogenicity of HA epitope as assessed in HLA-DR1 transgenic mice. Our study demonstrates for the first time that MHC conformational changes, revealed at molecular level, may influence the immunogenicity.