A multispecific antibody prevents immune escape and confers pan-SARS-CoV-2 neutralization.

Despite effective countermeasures, SARS-CoV-2 persists worldwide due to its ability to diversify and evade human immunity1. This evasion stems from amino-acid substitutions, particularly in the receptor-binding domain of the spike, that confer resistance to vaccines and antibodies 2-16. To constrain viral escape through resistance mutations, we combined antibody variable regions that recognize different receptor binding domain (RBD) sites17,18 into multispecific antibodies. Here, we describe multispecific antibodies, including a trispecific that prevented virus escape >3000-fold more potently than the most effective clinical antibody or mixtures of the parental antibodies. Despite being generated before the evolution of Omicron, this trispecific antibody potently neutralized all previous variants of concern and major Omicron variants, including the most recent BA.4/BA.5 strains at nanomolar concentrations. Negative stain electron microscopy revealed that synergistic neutralization was achieved by engaging different epitopes in specific orientations that facilitated inter-spike binding. An optimized trispecific antibody also protected Syrian hamsters against Omicron variants BA.1, BA.2 and BA.5, each of which uses different amino acid substitutions to mediate escape from therapeutic antibodies. Such multispecific antibodies decrease the likelihood of SARS-CoV-2 escape, simplify treatment, and maximize coverage, providing a strategy for universal antibody therapies that could help eliminate pandemic spread for this and other pathogens.

Immunogenicity and protective efficacy of RSV G central conserved domain vaccine with a prefusion nanoparticle.

Respiratory syncytial virus (RSV) G glycoprotein has recently reemerged as a vaccine antigen due to its ability to elicit potent neutralizing antibodies and ameliorate disease in animal models. Here we designed three constructs to display the G central conserved domain (Gcc) focused on inducing broad and potent neutralizing antibodies. One construct displaying Gcc from both RSV subgroups trimerized via a C-terminal foldon (Gcc-Foldon) was highly immunogenic in mice and in MIMIC, a pre-immune human in vitro model. To explore an optimal RSV vaccine, we combined the Gcc-Foldon antigen with a stabilized pre-fusion-F nanoparticle (pre-F-NP) as a bivalent vaccine and detected no antigenic interference between the two antigens in the MIMIC model. In RSV-primed macaques, the bivalent vaccine elicited potent humoral responses. Furthermore, both Gcc-Foldon and the bivalent vaccine conferred effective protection against RSV challenge in mice. This two-component vaccine could potentially provide effective protection against RSV infection in humans and warrants further clinical evaluation.

A bivalent Epstein-Barr virus vaccine induces neutralizing antibodies that block infection and confer immunity in humanized mice.

Epstein-Barr virus (EBV) is the major cause of infectious mononucleosis and is associated with several human cancers and, more recently, multiple sclerosis. Despite its prevalence and health impact, there are currently no vaccines or treatments. Four viral glycoproteins (gp), gp350 and gH/gL/gp42, mediate entry into the major sites of viral replication, B cells, and epithelial cells. Here, we designed a nanoparticle vaccine displaying these proteins and showed that it elicits potent neutralizing antibodies that protect against infection in vivo. We designed single-chain gH/gL and gH/gL/gp42 proteins that were each fused to bacterial ferritin to form a self-assembling nanoparticle. Structural analysis revealed that single-chain gH/gL and gH/gL/gp42 adopted a similar conformation to the wild-type proteins, and the protein spikes were observed by electron microscopy. Single-chain gH/gL or gH/gL/gp42 nanoparticle vaccines were constructed to ensure product homogeneity needed for clinical development. These vaccines elicited neutralizing antibodies in mice, ferrets, and nonhuman primates that inhibited EBV entry into both B cells and epithelial cells. When mixed with a previously reported gp350 nanoparticle vaccine, gp350D123, no immune competition was observed. To confirm its efficacy in vivo, humanized mice were challenged with EBV after passive transfer of IgG from mice vaccinated with control, gH/gL/gp42+gp350D123, or gH/gL+gp350D123 nanoparticles. Although all control animals were infected, only one mouse in each vaccine group that received immune IgG had detectable transient viremia. Furthermore, no EBV lymphomas were detected in immune animals. This bivalent EBV nanoparticle vaccine represents a promising candidate to prevent EBV infection and EBV-related malignancies in humans.

Broad neutralization of H1 and H3 viruses by adjuvanted influenza HA stem vaccines in nonhuman primates.

Seasonal influenza vaccines confer protection against specific viral strains but have restricted breadth that limits their protective efficacy. The H1 and H3 subtypes of influenza A virus cause most of the seasonal epidemics observed in humans and are the major drivers of influenza A virus-associated mortality. The consequences of pandemic spread of COVID-19 underscore the public health importance of prospective vaccine development. Here, we show that headless hemagglutinin (HA) stabilized-stem immunogens presented on ferritin nanoparticles elicit broadly neutralizing antibody (bnAb) responses to diverse H1 and H3 viruses in nonhuman primates (NHPs) when delivered with a squalene-based oil-in-water emulsion adjuvant, AF03. The neutralization potency and breadth of antibodies isolated from NHPs were comparable to human bnAbs and extended to mismatched heterosubtypic influenza viruses. Although NHPs lack the immunoglobulin germline VH1-69 residues associated with the most prevalent human stem-directed bnAbs, other gene families compensated to generate bnAbs. Isolation and structural analyses of vaccine-induced bnAbs revealed extensive interaction with the fusion peptide on the HA stem, which is essential for viral entry. Antibodies elicited by these headless HA stabilized-stem vaccines neutralized diverse H1 and H3 influenza viruses and shared a mode of recognition analogous to human bnAbs, suggesting that these vaccines have the potential to confer broadly protective immunity against diverse viruses responsible for seasonal and pandemic influenza infections in humans.

Evaluation of the respiratory syncytial virus G-directed neutralizing antibody response in the human airway epithelial cell model.

Human respiratory syncytial virus (RSV) is a major cause of serious respiratory tract infections in infants and the elderly. Recently it was shown that the RSV G glycoprotein mediates attachment to cells using CX3CR1 as a receptor, and that G-specific neutralizing antibodies can be detected using human airway epithelial (HAE) cell cultures. To investigate the contributions of G-specific antibodies to RSV neutralization, we performed HAE neutralization assays on sera from RSV G-immunized mice or RSV-infected infants. We confirmed that G-specific neutralization using serum from mice or humans could only be detected on HAE cultures. We also found that RSV G-specific antibodies in infants were either subgroup specific or cross-neutralizing. Altogether, our results suggest that G is an important target for generating neutralizing antibodies and would be beneficial to include in an RSV vaccine. Further, inclusion of G antigens from both RSV subgroups may enhance the vaccine cross protection potency.

Author Correction: Next-generation influenza vaccines: opportunities and challenges.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Design of a broadly reactive Lyme disease vaccine.

A growing global health concern, Lyme disease has become the most common tick-borne disease in the United States and Europe. Caused by the bacterial spirochete Borrelia burgdorferi sensu lato (sl), this disease can be debilitating if not treated promptly. Because diagnosis is challenging, prevention remains a priority; however, a previously licensed vaccine is no longer available to the public. Here, we designed a six component vaccine that elicits antibody (Ab) responses against all Borrelia strains that commonly cause Lyme disease in humans. The outer surface protein A (OspA) of Borrelia was fused to a bacterial ferritin to generate self-assembling nanoparticles. OspA-ferritin nanoparticles elicited durable high titer Ab responses to the seven major serotypes in mice and non-human primates at titers higher than a previously licensed vaccine. This response was durable in rhesus macaques for more than 6 months. Vaccination with adjuvanted OspA-ferritin nanoparticles stimulated protective immunity from both B. burgdorferi and B. afzelii infection in a tick-fed murine challenge model. This multivalent Lyme vaccine offers the potential to limit the spread of Lyme disease.

A respiratory syncytial virus (RSV) F protein nanoparticle vaccine focuses antibody responses to a conserved neutralization domain.

A stabilized form of the respiratory syncytial virus (RSV) fusion (F) protein has been explored as a vaccine to prevent viral infection because it presents several potent neutralizing epitopes. Here, we used a structure-based rational design to optimize antigen presentation and focus antibody (Ab) responses to key epitopes on the pre-fusion (pre-F) protein. This protein was fused to ferritin nanoparticles (pre-F-NP) and modified with glycans to mask nonneutralizing or poorly neutralizing epitopes to further focus the Ab response. The multimeric pre-F-NP elicited durable pre-F-specific Abs in nonhuman primates (NHPs) after >150 days and elicited potent neutralizing Ab (NAb) responses in mice and NHPs in vivo, as well as in human cells evaluated in the in vitro MIMIC system. This optimized pre-F-NP stimulated a more potent Ab response than a representative pre-F trimer, DS-Cav1. Collectively, this pre-F vaccine increased the generation of NAbs targeting the desired pre-F conformation, an attribute that facilitates the development of an effective RSV vaccine.

Next-generation influenza vaccines: opportunities and challenges.

Seasonal influenza vaccines lack efficacy against drifted or pandemic influenza strains. Developing improved vaccines that elicit broader immunity remains a public health priority. Immune responses to current vaccines focus on the haemagglutinin head domain, whereas next-generation vaccines target less variable virus structures, including the haemagglutinin stem. Strategies employed to improve vaccine efficacy involve using structure-based design and nanoparticle display to optimize the antigenicity and immunogenicity of target antigens; increasing the antigen dose; using novel adjuvants; stimulating cellular immunity; and targeting other viral proteins, including neuraminidase, matrix protein 2 or nucleoprotein. Improved understanding of influenza antigen structure and immunobiology is advancing novel vaccine candidates into human trials.

Comparison of adjuvants to optimize influenza neutralizing antibody responses.

Seasonal influenza vaccines represent a positive intervention to limit the spread of the virus and protect public health. Yet continual influenza evolution and its ability to evade immunity pose a constant threat. For these reasons, vaccines with improved potency and breadth of protection remain an important need. We previously developed a next-generation influenza vaccine that displays the trimeric influenza hemagglutinin (HA) on a ferritin nanoparticle (NP) to optimize its presentation. Similar to other vaccines, HA-nanoparticle vaccine efficacy is increased by the inclusion of adjuvants during immunization. To identify the optimal adjuvants to enhance influenza immunity, we systematically analyzed TLR agonists for their ability to elicit immune responses. HA-NPs were compatible with nearly all adjuvants tested, including TLR2, TLR4, TLR7/8, and TLR9 agonists, squalene oil-in-water mixtures, and STING agonists. In addition, we chemically conjugated TLR7/8 and TLR9 ligands directly to the HA-ferritin nanoparticle. These TLR agonist-conjugated nanoparticles induced stronger antibody responses than nanoparticles alone, which allowed the use of a 5000-fold-lower dose of adjuvant than traditional admixtures. One candidate, the oil-in-water adjuvant AF03, was also tested in non-human primates and showed strong induction of neutralizing responses against both matched and heterologous H1N1 viruses. These data suggest that AF03, along with certain TLR agonists, enhance strong neutralizing antibody responses following influenza vaccination and may improve the breadth, potency, and ultimately vaccine protection in humans.

Development of a Pan-H1 Influenza Vaccine.

The efficacy of current seasonal influenza vaccines varies greatly, depending on the match to circulating viruses. Although most vaccines elicit strain-specific responses, some present cross-reactive epitopes that elicit antibodies against diverse viruses and remain unchanged and effective for several years. To determine whether combinations of specific H1 hemagglutinin (HA) antigens stimulate immune responses that protect against diverse H1 influenza viruses, we evaluated the antibody responses elicited by HA-ferritin nanoparticles derived from six evolutionarily divergent H1 sequences and two computationally optimized broadly reactive antigen (COBRA) HA antigens. Humoral responses were assessed against a panel of 16 representative influenza virus strains from the past 80 years. HAs from the strains A/NewCaledonia/20/1999 (NC99), A/California/04/2009 (CA09), A/HongKong/117/1977 (HK77), COBRA X6, or P1 elicited neutralization against diverse strains, and a combination of three wild-type HA or two COBRA HA nanoparticles conferred significant additional breadth beyond that observed with any individual strain. Therefore, combinations of H1 HAs may constitute a pan-H1 influenza vaccine.IMPORTANCE Seasonal influenza vaccines elicit strain-specific immune responses designed to protect against circulating viruses. Because these vaccines often show limited efficacy, the search for a broadly protective seasonal vaccine remains a priority. Among different influenza virus subtypes, H1N1 has long been circulating in humans and has caused pandemic outbreaks. In order to assess the potential of a multivalent HA combination vaccine to improve the breadth of protection against divergent H1N1 viruses, HA-ferritin nanoparticles were made and evaluated in mice against a panel of historical and contemporary influenza virus strains. Trivalent combinations of H1 nanoparticles improved the breadth of immunity against divergent H1 influenza viruses.

Hemagglutinin-stem nanoparticles generate heterosubtypic influenza protection.

The antibody response to influenza is primarily focused on the head region of the hemagglutinin (HA) glycoprotein, which in turn undergoes antigenic drift, thus necessitating annual updates of influenza vaccines. In contrast, the immunogenically subdominant stem region of HA is highly conserved and recognized by antibodies capable of binding multiple HA subtypes. Here we report the structure-based development of an H1 HA stem-only immunogen that confers heterosubtypic protection in mice and ferrets. Six iterative cycles of structure-based design (Gen1-Gen6) yielded successive H1 HA stabilized-stem (HA-SS) immunogens that lack the immunodominant head domain. Antigenic characterization, determination of two HA-SS crystal structures in complex with stem-specific monoclonal antibodies and cryo-electron microscopy analysis of HA-SS on ferritin nanoparticles (H1-SS-np) confirmed the preservation of key structural elements. Vaccination of mice and ferrets with H1-SS-np elicited broadly cross-reactive antibodies that completely protected mice and partially protected ferrets against lethal heterosubtypic H5N1 influenza virus challenge despite the absence of detectable H5N1 neutralizing activity in vitro. Passive transfer of immunoglobulin from H1-SS-np-immunized mice to naive mice conferred protection against H5N1 challenge, indicating that vaccine-elicited HA stem-specific antibodies can protect against diverse group 1 influenza strains.

Flow cytometry reveals that H5N1 vaccination elicits cross-reactive stem-directed antibodies from multiple Ig heavy-chain lineages.

UNLABELLED: An understanding of the antigen-specific B-cell response to the influenza virus hemagglutinin (HA) is critical to the development of universal influenza vaccines, but it has not been possible to examine these cells directly because HA binds to sialic acid (SA) on most cell types. Here, we use structure-based modification of HA to isolate HA-specific B cells by flow cytometry and characterize the features of HA stem antibodies (Abs) required for their development. Incorporation of a previously described mutation (Y98F) to the receptor binding site (RBS) causes HA to bind only those B cells that express HA-specific Abs, but it does not bind nonspecifically to B cells, and this mutation has no effect on the binding of broadly neutralizing Abs to the RBS. To test the specificity of the Y98F mutation, we first demonstrated that previously described HA nanoparticles mediate hemagglutination and then determined that the Y98F mutation eliminates this activity. Cloning of immunoglobulin genes from HA-specific B cells isolated from a single human subject demonstrates that vaccination with H5N1 influenza virus can elicit B cells expressing stem monoclonal Abs (MAbs). Although these MAbs originated mostly from the IGHV1-69 germ line, a reasonable proportion derived from other genes. Analysis of stem Abs provides insight into the maturation pathways of IGVH1-69-derived stem Abs. Furthermore, this analysis shows that multiple non-IGHV1-69 stem Abs with a similar neutralizing breadth develop after vaccination in humans, suggesting that the HA stem response can be elicited in individuals with non-stem-reactive IGHV1-69 alleles. IMPORTANCE: Universal influenza vaccines would improve immune protection against infection and facilitate vaccine manufacturing and distribution. Flu vaccines stimulate B cells in the blood to produce antibodies that neutralize the virus. These antibodies target a protein on the surface of the virus called HA. Flu vaccines must be reformulated annually, because these antibodies are mostly specific to the viral strains used in the vaccine. But humans can produce broadly neutralizing antibodies. We sought to isolate B cells whose genes encode influenza virus antibodies from a patient vaccinated for avian influenza. To do so, we modified HA so it would bind only the desired cells. Sequencing the antibody genes of cells marked by this probe proved that the patient produced broadly neutralizing antibodies in response to the vaccine. Many sequences obtained had not been observed before. There are more ways to generate broadly neutralizing antibodies for influenza virus than previously thought.

Prime-boost interval matters: a randomized phase 1 study to identify the minimum interval necessary to observe the H5 DNA influenza vaccine priming effect.

BACKGROUND: H5 DNA priming was previously shown to improve the antibody response to influenza A(H5N1) monovalent inactivated vaccine (MIV) among individuals for whom there was a 24-week interval between prime and boost receipt. This study defines the shortest prime-boost interval associated with an improved response to MIV. METHODS: We administered H5 DNA followed by MIV at intervals of 4, 8, 12, 16, or 24 weeks and compared responses to that of 2 doses of MIV (prime-boost interval, 24 weeks). RESULTS: H5 DNA priming with an MIV boost ≥12 weeks later showed an improved response, with a positive hemagglutination inhibition (HAI) titer in 91% of recipients (geometric mean titer [GMT], 141-206), compared with 55%-70% of recipients with an H5 DNA and MIV prime-boost interval of ≤8 weeks (GMT, 51-70) and 44% with an MIV-MIV prime-boost interval of 24 weeks (GMT, 27). CONCLUSION: H5 DNA priming enhances antibody responses after an MIV boost when the prime-boost interval is 12-24 weeks. Clinical Trials Registration. NCT01086657.

Self-assembling influenza nanoparticle vaccines elicit broadly neutralizing H1N1 antibodies.

Influenza viruses pose a significant threat to the public and are a burden on global health systems. Each year, influenza vaccines must be rapidly produced to match circulating viruses, a process constrained by dated technology and vulnerable to unexpected strains emerging from humans and animal reservoirs. Here we use knowledge of protein structure to design self-assembling nanoparticles that elicit broader and more potent immunity than traditional influenza vaccines. The viral haemagglutinin was genetically fused to ferritin, a protein that naturally forms nanoparticles composed of 24 identical polypeptides. Haemagglutinin was inserted at the interface of adjacent subunits so that it spontaneously assembled and generated eight trimeric viral spikes on its surface. Immunization with this influenza nanoparticle vaccine elicited haemagglutination inhibition antibody titres more than tenfold higher than those from the licensed inactivated vaccine. Furthermore, it elicited neutralizing antibodies to two highly conserved vulnerable haemagglutinin structures that are targets of universal vaccines: the stem and the receptor binding site on the head. Antibodies elicited by a 1999 haemagglutinin-nanoparticle vaccine neutralized H1N1 viruses from 1934 to 2007 and protected ferrets from an unmatched 2007 H1N1 virus challenge. This structure-based, self-assembling synthetic nanoparticle vaccine improves the potency and breadth of influenza virus immunity, and it provides a foundation for building broader vaccine protection against emerging influenza viruses and other pathogens.

Elicitation of broadly neutralizing influenza antibodies in animals with previous influenza exposure.

The immune system responds to influenza infection by producing neutralizing antibodies to the viral surface protein, hemagglutinin (HA), which regularly changes its antigenic structure. Antibodies that target the highly conserved stem region of HA neutralize diverse influenza viruses and can be elicited through vaccination in animals and humans. Efforts to develop universal influenza vaccines have focused on strategies to elicit such antibodies; however, the concern has been raised that previous influenza immunity may abrogate the induction of such broadly protective antibodies. We show here that prime-boost immunization can induce broadly neutralizing antibody responses in influenza-immune mice and ferrets that were previously infected or vaccinated. HA stem-directed antibodies were elicited in mice primed with a DNA vaccine and boosted with inactivated vaccine from H1N1 A/New Caledonia/20/1999 (1999 NC) HA regardless of preexposure. Similarly, gene-based vaccination with replication-defective adenovirus 28 (rAd28) and 5 (rAd5) vectors encoding 1999 NC HA elicited stem-directed neutralizing antibodies and conferred protection against unmatched 1934 and 2007 H1N1 virus challenge in influenza-immune ferrets. Indeed, previous exposure to certain strains could enhance immunogenicity: The strongest HA stem-directed immune response was observed in ferrets previously infected with a divergent 1934 H1N1 virus. These findings suggest that broadly neutralizing antibodies against the conserved stem region of HA can be elicited through vaccination despite previous influenza exposure, which supports the feasibility of developing stem-directed universal influenza vaccines for humans.

Structural and genetic basis for development of broadly neutralizing influenza antibodies.

Influenza viruses take a yearly toll on human life despite efforts to contain them with seasonal vaccines. These viruses evade human immunity through the evolution of variants that resist neutralization. The identification of antibodies that recognize invariant structures on the influenza haemagglutinin (HA) protein have invigorated efforts to develop universal influenza vaccines. Specifically, antibodies to the highly conserved stem region of HA neutralize diverse viral subtypes. These antibodies largely derive from a specific antibody gene, heavy-chain variable region IGHV1-69, after limited affinity maturation from their germline ancestors, but how HA stimulates naive B cells to mature and induce protective immunity is unknown. To address this question, we analysed the structural and genetic basis for their engagement and maturation into broadly neutralizing antibodies. Here we show that the germline-encoded precursors of these antibodies act as functional B-cell antigen receptors (BCRs) that initiate subsequent affinity maturation. Neither the germline precursor of a prototypic antibody, CR6261 (ref. 3), nor those of two other natural human IGHV1-69 antibodies, bound HA as soluble immunoglobulin-G (IgG). However, all three IGHV1-69 precursors engaged HA when the antibody was expressed as cell surface IgM. HA triggered BCR-associated tyrosine kinase signalling by germline transmembrane IgM. Recognition and virus neutralization was dependent solely on the heavy chain, and affinity maturation of CR6261 required only seven amino acids in the complementarity-determining region (CDR) H1 and framework region 3 (FR3) to restore full activity. These findings provide insight into the initial events that lead to the generation of broadly neutralizing antibodies to influenza, informing the rational design of vaccines to elicit such antibodies and providing a model relevant to other infectious diseases, including human immunodeficiency virus/AIDS. The data further suggest that selected immunoglobulin genes recognize specific protein structural 'patterns' that provide a substrate for further affinity maturation.

Effect of priming with H1N1 influenza viruses of variable antigenic distances on challenge with 2009 pandemic H1N1 virus.

Compared to seasonal influenza viruses, the 2009 pandemic H1N1 (pH1N1) virus caused greater morbidity and mortality in children and young adults. People over 60 years of age showed a higher prevalence of cross-reactive pH1N1 antibodies, suggesting that they were previously exposed to an influenza virus or vaccine that was antigenically related to the pH1N1 virus. To define the basis for this cross-reactivity, ferrets were infected with H1N1 viruses of variable antigenic distance that circulated during different decades from the 1930s (Alaska/35), 1940s (Fort Monmouth/47), 1950s (Fort Warren/50), and 1990s (New Caledonia/99) and challenged with 2009 pH1N1 virus 6 weeks later. Ferrets primed with the homologous CA/09 or New Jersey/76 (NJ/76) virus served as a positive control, while the negative control was an influenza B virus that should not cross-protect against influenza A virus infection. Significant protection against challenge virus replication in the respiratory tract was observed in ferrets primed with AK/35, FM/47, and NJ/76; FW/50-primed ferrets showed reduced protection, and NC/99-primed ferrets were not protected. The hemagglutinins (HAs) of AK/35, FM/47, and FW/50 differ in the presence of glycosylation sites. We found that the loss of protective efficacy observed with FW/50 was associated with the presence of a specific glycosylation site. Our results suggest that changes in the HA occurred between 1947 and 1950, such that prior infection could no longer protect against 2009 pH1N1 infection. This provides a mechanistic understanding of the nature of serological cross-protection observed in people over 60 years of age during the 2009 H1N1 pandemic.

Immunogenicity and clinical protection against equine influenza by DNA vaccination of ponies.

Equine influenza A (H3N8) virus infection is a leading cause of respiratory disease in horses, resulting in widespread morbidity and economic losses. As with influenza in other species, equine influenza strains continuously mutate, often requiring the development of new vaccines. Current inactivated (killed) vaccines, while efficacious, only offer limited protection against diverse subtypes and require frequent boosts. Research into new vaccine technologies, including gene-based vaccines, aims to increase the neutralization potency, breadth, and duration of protective immunity. Here, we demonstrate that a DNA vaccine expressing the hemagglutinin protein of equine H3N8 influenza virus generates homologous and heterologous immune responses, and protects against clinical disease and viral replication by homologous H3N8 virus in horses. Furthermore, we demonstrate that needle-free delivery is as efficient and effective as conventional parenteral injection using a needle and syringe. These findings suggest that DNA vaccines offer a safe, effective, and promising alternative approach for veterinary vaccines against equine influenza.

Aerosolized adenovirus-vectored vaccine as an alternative vaccine delivery method.

Conventional parenteral injection of vaccines is limited in its ability to induce locally-produced immune responses in the respiratory tract, and has logistical disadvantages in widespread vaccine administration. Recent studies suggest that intranasal delivery or vaccination in the respiratory tract with recombinant viral vectors can enhance immunogenicity and protection against respiratory diseases such as influenza and tuberculosis, and can offer more broad-based generalized protection by eliciting durable mucosal immune responses. Controlled aerosolization is a method to minimize vaccine particle size and ensure delivery to the lower respiratory tract. Here, we characterize the dynamics of aerosolization and show the effects of vaccine concentration on particle size, vector viability, and the actual delivered dose of an aerosolized adenoviral vector. In addition, we demonstrate that aerosol delivery of a recombinant adenoviral vaccine encoding H1N1 hemagglutinin is immunogenic and protects ferrets against homologous viral challenge. Overall, aerosol delivery offers comparable protection to intramuscular injection, and represents an attractive vaccine delivery method for broad-based immunization campaigns.