Anti-hemagglutinin monomeric nanobody provides prophylactic immunity against H1 subtype influenza A viruses.

Influenza viruses constitute a major threat to human health globally. The viral surface glycoprotein hemagglutinin (HA) is the immunodominant antigen, contains the site for binding to the cellular receptor (RBS), and it is the major target of neutralizing antibody responses post-infection. We developed llama-derived single chain antibody fragments (VHHs) specific for type A influenza virus. Four VHHs were identified and further characterized. VHH D81 bound residues in the proximity of the C-terminal region of HA1 of H1 and H5 subtypes, and showed weak neutralizing activity, whereas VHH B33 bound residues in the proximity of the N-terminal region of the HA's stem domain (HA2) of H1, H5, and H9 subtypes, and showed no neutralizing activity. Of most relevance, VHHs E13 and G41 recognized highly conserved conformational epitopes on the H1 HA's globular domain (HA1) and showed high virus neutralizing activity (ranging between 0.94 to 0.01µM), when tested against several human H1N1 isolates. Additionally, E13 displayed abrogated virus replication of a panel of H1N1 strains spanning over 80 years of antigenic drift and isolated from human, avian, and swine origin. Interestingly, E13 conferred protection in vivo at a dose as low as 0.05 mg/kg. Mice treated with E13 intranasally resulted in undetectable virus challenge loads in the lungs at day 4 post-challenge. The transfer of sterilizing pan-H1 immunity, by a dose in the range of micrograms given intranasally, is of major significance for a monomeric VHH and supports the further development of E13 as an immunotherapeutic agent for the mitigation of influenza infections.

Sequence-Based Antigenic Analyses of H1 Swine Influenza A Viruses from Colombia (2008-2021) Reveals Temporal and Geographical Antigenic Variations.

Swine influenza is a respiratory disease that affects the pork industry and is a public health threat. It is caused by type A influenza virus (FLUAV), which continuously undergoes genetic and antigenic variations. A large amount of information regarding FLUAV in pigs is available worldwide, but it is limited in Latin America. The HA sequences of H1 subtype FLUAV-positive samples obtained from pigs in Colombia between 2008-2021 were analyzed using sequence-based antigenic cartography and N-Glycosylation analyses. Of the 12 predicted global antigenic groups, Colombia contained five: four corresponding to pandemic strains and one to the classical swine H1N1 clade. Circulation of these clusters was observed in some regions during specific years. Ca2 was the immunodominant epitope among Colombian viruses. The counts of N-Glycosylation motifs were associated with the antigenic cluster ranging from three to five. The results show for the first time the existence of antigenic diversity of FLUAV in Colombia and highlight the impact of spatial and temporal factors on this diversity. This study provides information about FLUAV variability in pigs under natural conditions in the absence of vaccination and emphasizes the need for surveillance of its phylogenetic and antigenic characteristics.

A(H3N2) antigenic variation of influenza is associated with low vaccine efficacy in the early 2018 influenza season in Mexico City.

OBJECTIVES: We evaluated the VE and the mutations of the viruses present in the Mexican population at the beginning of 2018. METHODS: We diagnosed influenza in outpatients with a high-performance Rapid Influenza Diagnostic Test (RIDT) qRT-PCR. Descriptive statistics were used to describe the study population, while the chi-square test was used to determine clinical variables. VE was analyzed through a negative test design. We sequenced the hemagglutinin (HA) gene, performed a phylogenetic analysis, and analyzed the nonsynonymous substitutions both in and outside antigenic sites. RESULTS: Of the 240 patients analyzed, 42.5% received the trivalent vaccine, and 37.5% were positive for influenza. The VE for the general population for any influenza virus type or subtype was 37.0%, while the VE for the predominant influenza A(H3N2) subtype was the lowest (19.7%). The phylogenetic analysis of HA showed the co-circulation of clades and subclades 3C.2a1, 3C.2a1b, 3C.2a2, 3C.2a2re, 3C.2a3, and 3C.3a with identities approximately 97-98% similar to the vaccine composition. CONCLUSION: Low VE was related to the co-circulation of multiple clades and subclades of influenza A(H3N2), with sufficient genetic and phenotypic distance to allow for the infection of vaccinated individuals.

Evolutionary Dynamics of Mexican Lineage H5N2 Avian Influenza Viruses.

We have demonstrated for the first time a comprehensive evolutionary analysis of the Mexican lineage H5N2 avian influenza virus (AIV) using complete genome sequences (n = 189), from its first isolation in 1993 until 2019. Our study showed that the Mexican lineage H5N2 AIV originated from the North American wild bird gene pool viruses around 1990 and is currently circulating in poultry populations of Mexico, the Dominican Republic, and Taiwan. Since the implementation of vaccination in 1995, the highly pathogenic AIV (HPAIV) H5N2 virus was eradicated from Mexican poultry in mid-1995. However, the low pathogenic AIV (LPAIV) H5N2 virus has continued to circulate in domestic poultry populations in Mexico, eventually evolving into five distinct clades. In the current study, we demonstrate that the evolution of Mexican lineage H5N2 AIVs involves gene reassortments and mutations gained over time. The current circulating Mexican lineage H5N2 AIVs are classified as LPAIV based on the amino acid sequences of the hemagglutinin (HA) protein cleavage site motif as well as the results of the intravenous pathogenicity index (IVPI). The immune pressure from vaccinations most likely has played a significant role in the positive selection of antigenic drift mutants within the Mexican H5N2 AIVs. Most of the identified substitutions in these viruses are located on the critical antigenic residues of the HA protein and as a result, might have contributed to vaccine failures. This study highlights and stresses the need for vaccine updates while emphasizing the importance of continued molecular monitoring of the HA protein for its antigenic changes compared to the vaccines used.

Characterization and use in neutralization assays of avian influenza codon-optimized H5 and H7 retroviral pseudotypes.

Influenza is a relevant problem for public and animal health, with a significant economic impact. In recent years, outbreaks of avian influenza virus have resulted in devastating losses in the poultry industry worldwide, and although its transmission to humans is very rare, there is always a potential risk for an even more severe outbreak. Currently, vaccination is considered the most effective tool for the control and prevention of influenza infections in both humans and animals. The maintenance of animal welfare and the successful implementation of animal health programs depend on the timely administration of vaccines, which must comply with quality specifications indicated by health authorities; for example, the capability to ensure a minimum antibody titer. The production of viral antigens used in these tests can pose a biosafety risk, and some viral strains can be difficult to grow. Therefore, new biotechnological alternatives are required to overcome these disadvantages. In this study, we produced pseudotypes carrying H5 and H7 hemagglutinins from lowly and highly pathogenic avian influenza viruses. These pseudotypes were used in neutralization assays to detect neutralizing antibodies in avian sera, which were confirmed positive by inhibition of the hemagglutination test. Our results showed that the pseudotype neutralization assay is a viable alternative for the detection of neutralizing antibodies, by demonstrating subtype specificity and requiring reduced biosafety requirements. Therefore, it represents a versatile platform that can facilitate technology transfer protocols between laboratories, and an immediate application in serological tools for quality control of veterinary vaccines against avian influenza.

Influence of adjuvants on the amount, specificity and functional activity of antibody response to human influenza vaccine in mice.

It's been almost a century since immunologists started using adjuvants as tools to develop more effective vaccines. Despite the rising number of adjuvanted vaccines in the last decades, we still lack knowledge of the adjuvants' effects on antibody response. This study was aimed to test the effect of immunizing mice with the human Inactivated Influenza vaccine (IIV), either alone or combined with different widely used adjuvants on the specific antibody response induced. Differential levels of IgM and IgG subclasses were found with the different adjuvants tested. Higher levels of antibodies did not always correspond with a higher efficacy to interfere with the virus infectivity. Differences in neutralization properties are possibly mediated by the specificity of the repertoire of antibodies induced. The repertoire was studied using a phage display 7-mer peptide library to screen for epitopes/mimotopes recognized by serum pools from vaccinated mice. The selected phage clones included peptides that corresponded to conformational mimotopes since they have no homology with lineal sequences of the Influenza strains' proteins. Five peptides were identified as recognized by sera from mice immunized with the IIV vaccine alone, including peptides from the hemagglutinin stalk domain, and by sera from mice immunized with the vaccine plus the different adjuvants employed. Adjuvants elicited a more diverse repertoire of epitope-recognizing antibodies that recognized epitopes of the HA recombinant globular head. Mimotopes were theoretically located at the neutralizing antigenic sites of the globular head of Influenza A H1N1pdm09, Influenza A H3N2, and Influenza B hemagglutinin. This study illustrates how different adjuvants can modify the extent and quality of humoral immunity against the IIV vaccine and the effectiveness of vaccination.

Symptoms, Infection Duration, and Hemagglutinin Inhibition Antibody Response in Influenza A Infections.

BACKGROUND: Many influenza studies assume that symptomatic and asymptomatic cases have equivalent antibody responses. METHODS: This study examines the relationship between influenza symptoms and serological response. Influenza-positive index cases and household members in Managua, Nicaragua, during 2012-2017 were categorized by symptom status. RESULTS: Antibody response was assessed using hemagglutination inhibition assays (HAI). Among 510 cases, 74.5% had ≥4-fold increase in HAI antibodies, and 75.3% had febrile illness. In a logistic regression model, febrile cases had 2.17 times higher odds of a ≥4-fold titer rise compared to asymptomatic cases (95% confidence interval, 1.02-4.64). CONCLUSIONS: Studies relying on serological assays may not generalize to asymptomatic infections.

Equine-Like H3 Avian Influenza Viruses in Wild Birds, Chile.

Since their discovery in the United States in 1963, outbreaks of infection with equine influenza virus (H3N8) have been associated with serious respiratory disease in horses worldwide. Genomic analysis suggests that equine H3 viruses are of an avian lineage, likely originating in wild birds. Equine-like internal genes have been identified in avian influenza viruses isolated from wild birds in the Southern Cone of South America. However, an equine-like H3 hemagglutinin has not been identified. We isolated 6 distinct H3 viruses from wild birds in Chile that have hemagglutinin, nucleoprotein, nonstructural protein 1, and polymerase acidic genes with high nucleotide homology to the 1963 H3N8 equine influenza virus lineage. Despite the nucleotide similarity, viruses from Chile were antigenically more closely related to avian viruses and transmitted effectively in chickens, suggesting adaptation to the avian host. These studies provide the initial demonstration that equine-like H3 hemagglutinin continues to circulate in a wild bird reservoir.

Serological evidence of the infection of H7 virus and the co-infection of H7 and H9 viruses in farmed fur-bearing animals in eastern China.

Avian influenza virus (AIV) usually infects wild birds and domestic poultry; however, this virus could be transmitted to mammals and humans. The previous studies reported that the farmed mink could be infected with the H5 AIV and H9 AIV, indicating that the farmed fur-bearing animals may be susceptible to AIV. Here, we report the serological evidence of infection of H7 AIV and co-infection of H7 and H9 AIV in healthy framed fur-bearing animals. We collected serum specimens from healthy farmed fur-bearing animals (farmed mink and farmed fox) and make an investigation of serological surveillance of clade 2.3.2 H5 AIV, clade 7.2 H5 AIV, clade 2.3.4.4 H5 AIV, H7 AIV, and H9 AIV. We did not find the hemagglutination inhibition (HI) antibodies against clade 2.3.2 H5 AIV, clade 7.2 H5 AIV, or clade 2.3.4.4 H5 AIV in the serum specimens of farmed fur-bearing animals. However, we found that both farmed mink and farmed fox possess HI antibodies against H7 AIV or H9 AIV; furthermore, we found that some serum specimens possess both anti-H7 AIV antibodies and anti-H9 AIV HI antibodies, suggesting that one farmed fur-bearing animal can be infected with two different subtype AIVs and may play an important role in the reassortment course of the novel avian influenza viruses. Taken together, our data suggested that the enhanced surveillance of AIV in farmed fur-bearing animals and humans or animals in close contact with them is needed.

Antigenic characterization of novel H1 influenza A viruses in swine.

Novel H1N2 influenza A viruses (IAVs) in swine have been identified in Chile co-circulating with pandemic H1N1 2009-like (A(H1N1)pdm09-like) viruses. The objective of this study was to characterize antigenically the swine H1 IAVs circulating in Chile. Genetic analysis based on the HA1 domain and antigenic analysis by hemagglutination inhibition assay were carried out. Three antigenic clusters were identified, named Chilean H1 A (ChH1A), Chilean H1 B (ChH1B), and A(H1N1)pdm09-like. The antigenic sites of ChH1A and ChH1B strains were 10-60% distant from those of commercial vaccine strains at the amino acid sequence level. Antigenic variants were identified within the clusters ChH1A and A(H1N1)pdm09-like. Substitutions in the main antigenic sites (E153G in Sa, Q193H in Sb, D168N in Ca1, P137S in Ca2, and F71L in Cb) were detected in variants from the ChH1A cluster, whereas only a single substitution in antigenic site Sa (G155E) was detected in variants from A(H1N1)pdm09-like cluster, which confirms the importance to carrying out antigenic analyses in addition to genetic analyses to evaluate control measures such as vaccination. These results highlight the need to update vaccines for swine in Chile and the importance of continued surveillance to determine the onward transmission of antigenic variants in Chilean pig populations.

Pre-existing Antineuraminidase Antibodies Are Associated With Shortened Duration of Influenza A(H1N1)pdm Virus Shedding and Illness in Naturally Infected Adults.

BACKGROUND: Influenza causes a substantial burden worldwide, and current seasonal influenza vaccine has suboptimal effectiveness. To develop better, more broadly protective vaccines, a more thorough understanding is needed of how antibodies that target the influenza virus surface antigens, hemagglutinin (HA) (including head and stalk regions) and neuraminidase (NA), impact influenza illness and virus transmission. METHODS: We used a case-ascertained, community-based study of household influenza virus transmission set in Managua, Nicaragua. Using data from 170 reverse transcriptase-polymerase chain reaction (RT-PCR)-confirmed influenza virus A(H1N1)pdm infections and 45 household members with serologically confirmed infection, we examined the association of pre-existing NA, hemagglutination inhibiting, and HA stalk antibody levels and influenza viral shedding and disease duration using accelerated failure time models. RESULTS: Among RT-PCR-confirmed infections in adults, pre-existing anti-NA antibody levels ≥40 were associated with a 69% (95% confidence interval [CI], 34-85%) shortened shedding duration (mean, 1.0 vs 3.2 days). Neuraminidase antibody levels ≥80 were associated with further shortened shedding and significantly shortened symptom duration (influenza-like illness, 82%; 95% CI, 39-95%). Among RT-PCR-confirmed infections in children, hemagglutination inhibition titers ≥1:20 were associated with a 32% (95% CI, 13-47%) shortened shedding duration (mean, 3.9 vs 6.0 days). CONCLUSIONS: Our results suggest that anti-NA antibodies play a large role in reducing influenza illness duration in adults and may impact transmission, most clearly among adults. Neuraminidase should be considered as an additional target in next-generation influenza virus vaccine development.We found that antibodies against neuraminidase were associated with significantly shortened viral shedding, and among adults they were also associated with shortened symptom duration. These results support neuraminidase as a potential target of next-generation influenza virus vaccines.

Protection of White Leghorn chickens by recombinant fowlpox vector vaccine with an updated H5 insert against Mexican H5N2 avian influenza viruses.

Despite decades of vaccination, surveillance, and biosecurity measures, H5N2 low pathogenicity avian influenza (LPAI) virus infections continue in Mexico and neighboring countries. One explanation for tenacity of H5N2 LPAI in Mexico is the antigenic divergence of circulating field viruses compared to licensed vaccines due to antigenic drift. Our phylogenetic analysis indicates that the H5N2 LPAI viruses circulating in Mexico and neighboring countries since 1994 have undergone antigenic drift away from vaccine seed strains. Here we evaluated the efficacy of a new recombinant fowlpox virus vector containing an updated H5 insert (rFPV-H5/2016), more relevant to the current strains circulating in Mexico. We tested the vaccine efficacy against a closely related subcluster 4 Mexican H5N2 LPAI (2010 H5/LP) virus and the historic H5N2 HPAI (1995 H5/HP) virus in White Leghorn chickens. The rFPV-H5/2016 vaccine provided hemagglutinin inhibition (HI) titers pre-challenge against viral antigens from both challenge viruses in almost 100% of the immunized birds, with no differences in number of birds seroconverting or HI titers among all tested doses (1.5, 2.0, and 3.1 log10 mean tissue culture infectious doses/bird). The vaccine conferred 100% clinical protection and a significant decrease in oral and cloacal virus shedding from 1995 H5/HP virus challenged birds when compared to the sham controls at all tested doses. Virus shedding titers from vaccinated 2010 H5/LP virus challenged birds significantly decreased compared to sham birds especially at earlier time points. Our results confirm the efficacy of the new rFPV-H5/2016 against antigenic drift of LPAI virus in Mexico and suggest that this vaccine would be a good candidate, likely as a primer in a prime-boost vaccination program.

An Inexpensive and Accurate Reverse Transcription-PCR-Melting Temperature Analysis Assay for Real-Time Influenza Virus B Lineage Discrimination.

In this work, we describe a SYBR-Green one-step reverse transcription-PCR protocol coupled with a melting temperature analysis (RT-PCR-Tm ), which allows the discrimination of influenza B lineages Yamagata and Victoria. The assay is performed using a regular real-time thermocycler and is based on differences in melting temperature (Tm ) of a 131-bp amplicon, obtained from a conserved region of hemagglutinin gene. A total of 410 samples collected during the 2004, 2008, and 2010-2017 influenza seasons in Brazil were tested, and the lineages were correctly characterized using their melting profiles. The temperature range is significantly different between both lineages throughout the time (Mann-Whitney test; P < 0.0001, confidence interval = 95%), and the Tm is not affected by viral load (Spearman correlation test; r = 0.287, P = 2.245 × 10-9). The simplicity and cost-effectiveness of this protocol make it an option for influenza B lineage surveillance worldwide.

Amino acid changes in HA and determinants of pathogenicity associated with influenza virus A H1N1pdm09 during the winter seasons 2015-2016 and 2016-2017 in Mexico.

Biennial H1N1pdm09 influenza A virus (IAV) epidemics have been associated with major severity of respiratory disease in Mexico. Atypically and in contrast with what happened in USA, Canada and Europe during 2017, an increase of infections due to the H1N1pdm09 pandemic virus instead of H3N2 was observed. In order to determine the viral contribution to severe acute respiratory disease, we characterized the pathogenicity determinants of IAV in Mexico during the 2015-2016 and 2016-2017 seasons. The RNA segments of 20 IAV samples were sequenced by NGS platform and phylogenetic analysis was conducted. The analysis of the hemagglutinin (HA) sequences established that all virus samples, except one, belong to clade (6B.1). The IAVs presented the substitution S162 N, which introduces a new glycosylation site in the hemagglutinin. We also found the D222 G substitution, which has been associated with a higher tropism towards the lower respiratory tract, and a non-reported insertion of one Ile in NS1 (Ile113). The IAVs from 2016 to 2017 in Mexico belong to the new clade 6B.1. The new glycosylation site in HA (S162 N) is a major change that may affect the efficacy of the current vaccine. We detected in several patients pathogenicity determinants associated with the severity of the respiratory disease.

Novel correlates of protection against pandemic H1N1 influenza A virus infection.

Influenza viruses remain a severe threat to human health, causing up to 650,000 deaths annually1,2. Seasonal influenza virus vaccines can prevent infection, but are rendered ineffective by antigenic drift. To provide improved protection from infection, novel influenza virus vaccines that target the conserved epitopes of influenza viruses, specifically those in the hemagglutinin stalk and neuraminidase, are currently being developed3. Antibodies against the hemagglutinin stalk confer protection in animal studies4-6. However, no data exist on natural infections in humans, and these antibodies do not show activity in the hemagglutination inhibition assay, the hemagglutination inhibition titer being the current correlate of protection against influenza virus infection7-9. While previous studies have investigated the protective effect of cellular immune responses and neuraminidase-inhibiting antibodies, additional serological correlates of protection from infection could aid the development of broadly protective or universal influenza virus vaccines10-13. To address this gap, we performed a household transmission study to identify alternative correlates of protection from infection and disease in naturally exposed individuals. Using this study, we determined 50% protective titers and levels for hemagglutination inhibition, full-length hemagglutinin, neuraminidase and hemagglutinin stalk-specific antibodies. Further, we found that hemagglutinin stalk antibodies independently correlated with protection from influenza virus infection.

Loss of Fitness of Mexican H7N3 Highly Pathogenic Avian Influenza Virus in Mallards after Circulating in Chickens.

Outbreaks of highly pathogenic avian influenza (HPAI) virus subtype H7N3 have been occurring in commercial chickens in Mexico since its first introduction in 2012. In order to determine changes in virus pathogenicity and adaptation in avian species, three H7N3 HPAI viruses from 2012, 2015, and 2016 were evaluated in chickens and mallards. All three viruses caused high mortality in chickens when given at medium to high doses and replicated similarly. No mortality or clinical signs and similar infectivity were observed in mallards inoculated with the 2012 and 2016 viruses. However, the 2012 H7N3 HPAI virus replicated well in mallards and transmitted to contacts, whereas the 2016 virus replicated poorly and did not transmit to contacts, which indicates that the 2016 virus is less adapted to mallards. In vitro, the 2016 virus grew slower and to lower titers than did the 2012 virus in duck fibroblast cells. Full-genome sequencing showed 115 amino acid differences between the 2012 and the 2016 viruses, with some of these changes previously associated with changes in replication in avian species, including hemagglutinin (HA) A125T, nucleoprotein (NP) M105V, and NP S377N. In conclusion, as the Mexican H7N3 HPAI virus has passaged through large populations of chickens in a span of several years and has retained its high pathogenicity for chickens, it has decreased in fitness in mallards, which could limit the potential spread of this HPAI virus by waterfowl.IMPORTANCE Not much is known about changes in host adaptation of avian influenza (AI) viruses in birds after long-term circulation in chickens or other terrestrial poultry. Although the origin of AI viruses affecting poultry is wild aquatic birds, the role of these birds in further dispersal of poultry-adapted AI viruses is not clear. Previously, we showed that HPAI viruses isolated early from poultry outbreaks could still infect and transmit well in mallards. In this study, we demonstrate that the Mexican H7N3 HPAI virus after four years of circulation in chickens replicates poorly and does not transmit in mallards but remains highly pathogenic in chickens. This information on changes in host adaptation is important for understanding the epidemiology of AI viruses and the role that wild waterfowl may play in disseminating viruses adapted to terrestrial poultry.

Efficacy of novel recombinant fowlpox vaccine against recent Mexican H7N3 highly pathogenic avian influenza virus.

Since 2012, H7N3 highly pathogenic avian influenza (HPAI) has produced negative economic and animal welfare impacts on poultry in central Mexico. In the present study, chickens were vaccinated with two different recombinant fowlpox virus vaccines (rFPV-H7/3002 with 2015 H7 hemagglutinin [HA] gene insert, and rFPV-H7/2155 with 2002 H7 HA gene insert), and were then challenged three weeks later with H7N3 HPAI virus (A/chicken/Jalisco/CPA-37905/2015). The rFPV-H7/3002 vaccine conferred 100% protection against mortality and morbidity, and significantly reduced virus shed titers from the respiratory and gastrointestinal tracts. In contrast, 100% of sham and rFPV-H7/2155 vaccinated birds shed virus at higher titers and died within 4 days. Pre- (15/20) and post- (20/20) challenge serum of birds vaccinated with rFPV-H7/3002 had antibodies detectable by hemagglutination inhibition (HI) assay using challenge virus antigen. However, only a few birds (3/20) in the rFPV-H7/2155 vaccinated group had antibodies that reacted against the challenge strain but all birds had antibodies that reacted against the homologous vaccine antigen (A/turkey/Virginia/SEP-66/2002) (20/20). One possible explanation for differences in vaccines efficacy is the antigenic drift between circulating viruses and vaccines. Molecular analysis demonstrated that the Mexican H7N3 strains have continued to rapidly evolve since 2012. In addition, we identified in silico three potential new N-glycosylation sites on the globular head of the H7 HA of A/chicken/Jalisco/CPA-37905/2015 challenge virus, which were absent in 2012 H7N3 outbreak virus. Our results suggested that mutations in the HA antigenic sites including increased glycosylation sites, accumulated in the new circulating Mexican H7 HPAIV strains, altered the recognition of neutralizing antibodies from the older vaccine strain rFPV-H7/2155. Therefore, the protective efficacy of novel rFPV-H7/3002 against recent outbreak Mexican H7N3 HPAIV confirms the importance of frequent updating of vaccines seed strains for long-term effective control of H7 HPAI virus.

Efficient oral vaccination by bioengineering virus-like particles with protozoan surface proteins.

Intestinal and free-living protozoa, such as Giardia lamblia, express a dense coat of variant-specific surface proteins (VSPs) on trophozoites that protects the parasite inside the host's intestine. Here we show that VSPs not only are resistant to proteolytic digestion and extreme pH and temperatures but also stimulate host innate immune responses in a TLR-4 dependent manner. We show that these properties can be exploited to both protect and adjuvant vaccine antigens for oral administration. Chimeric Virus-like Particles (VLPs) decorated with VSPs and expressing model surface antigens, such as influenza virus hemagglutinin (HA) and neuraminidase (NA), are protected from degradation and activate antigen presenting cells in vitro. Orally administered VSP-pseudotyped VLPs, but not plain VLPs, generate robust immune responses that protect mice from influenza infection and HA-expressing tumors. This versatile vaccine platform has the attributes to meet the ultimate challenge of generating safe, stable and efficient oral vaccines.

Exploring the nature of the H-bonds between the human class II MHC protein, HLA-DR1 (DRB*0101) and the influenza virus hemagglutinin peptide, HA306-318, using the quantum theory of atoms in molecules.

The nature of the H-bonds between the human protein HLA-DR1 (DRB*0101) and the hemagglutinin peptide HA306-318 has been studied using the Quantum Theory of Atoms in Molecules for the first time. We have found four H-bond groups: one conventional CO··HN bond group and three nonconventional CO··HC, π··HC involving aromatic rings and HN··HCaliphatic groups. The calculated electron density at the determined H-bond critical points suggests the follow protein pocket binding trend: P1 (2,311) >> P9 (1.109) > P4 (0.950) > P6 (0.553) > P7 (0.213) which agrees and reveal the nature of experimental findings, showing that P1 produces by a long way the strongest binding of the HLA-DR1 human protein molecule with the peptide backbone as consequence of the vast number of H-bonds in the P1 area and at the same time the largest specific binding of the peptide Tyr308 residue with aromatic residues located at the binding groove floor. The present results suggest the topological analysis of the electronic density as a valuable tool that allows a non-arbitrary partition of the pockets binding energy via the calculated electron density at the determined critical points.