SARS-CoV-2 Spike Protein-Expressing Enterococcus for Oral Vaccination: Immunogenicity and Protection.

The declaration of the conclusion of the COVID-19 pandemic notwithstanding, coronavirus remains prevalent in circulation, and the potential emergence of novel variants of concern introduces the possibility of new outbreaks. Moreover, it is not clear how quickly and to what extent the effectiveness of vaccination will decline as the virus continues to mutate. One possible solution to combat the rapidly mutating coronavirus is the creation of safe vaccine platforms that can be rapidly adapted to deliver new, specific antigens in response to viral mutations. Recombinant probiotic microorganisms that can produce viral antigens by inserting specific viral DNA fragments into their genome show promise as a platform and vector for mucosal vaccine antigen delivery. The authors of this study have developed a convenient and universal technique for inserting the DNA sequences of pathogenic bacteria and viruses into the gene that encodes the pili protein of the probiotic strain E. faecium L3. The paper presents data on the immunogenic properties of two E. faecium L3 vaccine strains, which produce two different fragments of the coronavirus S1 protein, and provides an assessment of the protective efficacy of these oral vaccines against coronavirus infection in Syrian hamsters.

Longitudinal Analysis of Neuraminidase and Hemagglutinin Antibodies to Influenza A Viruses after Immunization with Seasonal Inactivated Influenza Vaccines.

Neuraminidase (NA)-based immunity could reduce the harmful impact of novel antigenic variants of influenza viruses. The detection of neuraminidase-inhibiting (NI) antibodies in parallel with anti-hemagglutinin (HA) antibodies may enhance research on the immunogenicity and duration of antibody responses to influenza vaccines. To assess anti-NA antibodies after vaccination with seasonal inactivated influenza vaccines, we used the enzyme-linked lectin assay, and anti-HA antibodies were detected in the hemagglutination inhibition assay. The dynamics of the anti-NA antibody response differed depending on the virus subtype: antibodies to A/H3N2 virus neuraminidase increased later than antibodies to A/H1N1pdm09 subtype neuraminidase and persisted longer. In contrast to HA antibodies, the fold increase in antibody titers to NA after vaccination poorly depended on the preexisting level. At the same time, NA antibody levels after vaccination directly correlated with titers before vaccination. A difference was found in response to NA antigen between split and subunit-adjuvanted vaccines and in NA functional activity in the vaccine formulations.

Pilot Study Results on Antibodies to the S- and N-Proteins of SARS-CoV-2 in Paired Sera from COVID-19 Patients with Varying Severity.

In this retrospective cohort study, we investigated the formation of individual classes of antibodies to SARS-CoV-2 in archived serial sera from hospitalized patients with the medium-severe (n = 17) and severe COVID-19 (n = 11). The serum/plasma samples were studied for the presence of IgG, IgM and IgA antibodies to the recombinant S- and N-proteins of SARS-CoV-2. By the 7th day of hospitalization, an IgG increase was observed in patients both with a positive PCR test and without PCR confirmation of SARS-CoV-2 infection. Significant increases in the anti-spike IgG levels were noted only in moderate COVID-19. The four-fold increase of IgM to N-protein was obtained more often in the groups with mild and moderate infections. The IgA levels decreased during the infection to both the S- and N-proteins, and the most pronounced decrease was in the severe COVID-19 patients. The serum IgG to S-protein one week after hospitalization demonstrated a high-power relationship (rs = 0.75) with the level of RBD antibodies. There was a medium strength relationship between the levels of CRP and IgG (rs = 0.43). Thus, in patients with acute COVID-19, an increase in antibodies can develop as early as 1 week of hospital stay. The SARS-CoV-2 antibody conversions may confirm SARS-CoV-2 infection in PCR-negative patients.

Establishment of a Pseudovirus Platform for Neuraminidase Inhibiting Antibody Analysis.

Neuraminidase (NA)-based immunity to influenza can be useful for protecting against novel antigenic variants. To develop safe and effective tools to assess NA-based immunity, we generated a baculovirus-based pseudotyped virus, N1-Bac, that expresses the full-length NA of the influenza A/California/07/2009 (H1N1)pdm09 strain. We evaluated the level of NA-inhibiting (NI) antibodies in the paired blood sera of influenza patients by means of an enzyme-linked lectin assay (ELLA) using the influenza virus or N1-Bac. Additionally, we evaluated the level of NA antibodies by means of the enzyme-linked immunosorbent assay (ELISA) with an N1-expressing Sf21 culture. We detected a strong correlation between our results from using the influenza virus and NA-Bac pseudoviruses to detect NI antibodies and a medium-strong correlation between NI antibodies and NA antibodies determined by an N1-cell ELISA, indicating that baculovirus-based platforms can be successfully used to evaluate NI or NA antibodies. Furthermore, animal experiments showed that immunization with N1-Bac protected against infection with a drift variant of the A/H1N1pdm09 influenza virus. Our results demonstrate that recombinant baculovirus can be an effective influenza pseudotype to evaluate influenza serologic immunity and protect against influenza virus infection.

Evaluation of Immune Response to Mucosal Immunization with an Oral Probiotic-Based Vaccine in Mice: Potential for Prime-Boost Immunization against SARS-CoV-2.

Following the conclusion of the COVID-19 pandemic, the persistent genetic variability in the virus and its ongoing circulation within the global population necessitate the enhancement of existing preventive vaccines and the development of novel ones. A while back, we engineered an orally administered probiotic-based vaccine, L3-SARS, by integrating a gene fragment that encodes the spike protein S of the SARS-CoV-2 virus into the genome of the probiotic strain E. faecium L3, inducing the expression of viral antigen on the surface of bacteria. Previous studies demonstrated the efficacy of this vaccine candidate in providing protection against the virus in Syrian hamsters. In this present study, utilizing laboratory mice, we assess the immune response subsequent to immunization via the gastrointestinal mucosa and discuss its potential as an initial phase in a two-stage vaccination strategy. Our findings indicate that the oral administration of L3-SARS elicits an adaptive immune response in mice. Pre-immunization with L3-SARS enhances and prolongs the humoral immune response following a single subcutaneous immunization with a recombinant S-protein analogous to the S-insert of the coronavirus in Enterococcus faecium L3.

Antigenic Characterization of Neuraminidase of Influenza A/H7N9 Viruses Isolated in Different Years.

Influenza outbreaks caused by A/H7N9 viruses have occurred since 2013. After 2016, A/H7N9 influenza viruses underwent evolutionary changes. In this study, we examined the antigenic properties of influenza neuraminidase (NA) of A/H7N9 viruses as part of a live influenza vaccine (LAIV). It was shown that neuraminidase inhibiting (NI) antibodies obtained after A/Anhui/1/2013(H7N9)-based LAIV vaccination did not inhibit A/Hong Kong/125/2017(H7N9) NA and vice versa. The A/Hong Kong/125/2017(H7N9)-based LAIV elicited higher levels of NI antibodies compared to the A/Anhui/1/2013(H7N9)-based LAIV after two doses. Thelow degree of coincidence of the antibody response to hemagglutinin (HA) and NA after LAIV vaccination allows us to consider an enzyme-linked lectin assay (ELLA) as an additional measure for assessing the immunogenicity of influenza vaccines. In mice, N9-reactive monoclonal antibodies (mABs) for the A/environment/Shanghai/RL01/2013(H7N9) influenza virus partially protected against lung infection from the A/Guangdong/17SF003/2016 IDCDC-RG56N(H7N9) virus, thus showing the cross-protective properties of monoclonal antibodies against the drift variant.

Live Influenza Vaccine Provides Early Protection against Homologous and Heterologous Influenza and May Prevent Post-Influenza Pneumococcal Infections in Mice.

Influenza and S. pneumoniae infections are a significant cause of morbidity and mortality worldwide. Intranasal live influenza vaccine (LAIV) may prevent influenza-related bacterial complications. The objectives of the study are to estimate resistance against early influenza infection and post-influenza pneumococcal pneumonia after LAIV in mice. Mice were administered intranasally the monovalent LAIV A/17/Mallard Netherlands/00/95(H7N3), A/17/South Africa/2013/01(H1N1)pdm09 or trivalent LAIV 2017-2018 years of formulation containing A/17/New York/15/5364(H1N1)pdm09 vaccine strain. LAIV demonstrated early protection against homologous and heterologous infections with A/South Africa/3626/2013 (H1N1) pdm09 influenza virus on day six, following immunization. Following boost immunization, trivalent LAIV demonstrated a pronounced protective effect both in terms of lethality and pneumococcal lung infection when S. pneumoniae infection was performed three days after the onset of influenza infection. Conclusion: LAIV provides early protection against homologous and heterologous viral infections and has a protective effect against post-influenza pneumococcal infection. These data suggest that the intranasal administration of LAIV may be useful during the cycle of circulation not only of influenza viruses, but also of other causative agents of acute respiratory infections.

Study of Antibodies to Influenza Neuraminidase N2.

Humoral immunity to influenza neuraminidase (NA) was evaluated among different groups of people including patients with acute influenza infection and healthy people in different age groups using an enzyme linked lectin assay (ELLA). The amino acid composition of NA of seasonal influenza viruses A/Victoria/361/2011(H3N2) and A/Hong Kong/4801/2014(H3N2) differed by 2%, while cross-reacting neuraminidase-inhibiting (NI) antibodies to them in the same serum samples were detected in 10% of cases. Middle-aged patients born from 1977 to 2000 had a high level of hemagglutination-inhibiting (HI) antibodies to A/Hong Kong/4801/2014(H3N2), but almost no NI antibodies, which may indicate that in the case of a change in the hemagglutinin (HA) subtype, this age group will be susceptible to influenza A/H3N2 viruses. Therefore, it could mean there is a need for priority vaccination of this age group with a vaccine against the appropriate strain. It was shown that after intranasal administration of live influenza vaccine (LAIV) for the 2017-2018 season, serum antibody response was not lower compared to that during natural infection. In older people, antibodies to archival A/H2N2 viruses were detected more often than to modern A/H3N2. Since the conversion of antibodies to HA and NA often did not coincide, antibodies to NA can serve as an additional criterion for assessing the immunogenicity of influenza vaccines.

Associated virus-bacterial vaccine based on seasonal LAIV and S. pneumoniae chimeric peptide provide protection against post-influenza pneumococcal infection in mouse model.

Severe influenza complications are often caused by Streptococcus pneumoniae infection, which presents the most common cause of community-acquired pneumonia. We evaluated in a mouse model an associated virus-bacterial vaccine based on seasonal live influenza vaccines (LAIV) and S. pneumoniae chimeric protein comprising flagellin (PSPF). Intranasal immunization of mice with a complex of trivalent LAIV and PSPF caused an increased release of early cytokines in the lungs of mice. The immunogenicity of LAIV and PSPF in the associated vaccine composition was sometimes decreased compared to each vaccine preparation alone. Nevertheless, only vaccination of mice with LAIV+PSPF significantly reduced lethality and the bacterial load in the lungs in a model of post-influenza bacterial pneumonia. The study of the interactions of influenza viruses with bacterial peptides is important during the development of associated virus-bacterial vaccines intended for the prevention of severe post-influenza bacterial complications.

Developing a Live Probiotic Vaccine Based on the Enterococcus faecium L3 Strain Expressing Influenza Neuraminidase.

Probiotic microorganisms are currently considered as a promising platform for the development of recombinant vaccines expressing foreign antigens. In this study, we generated and evaluated the live mucosal recombinant vaccine by integrating genes encoding influenza virus neuraminidase (NA) of the N2 subtype into the DNA of the probiotic strain Enterococcus faecium L3 (L3). We confirmed NA expression in the pili of L3 using immune electron microscopy. Mice were fed with a probiotic vaccine containing the NA gene (L3-NA) or pure L3. Oral administration of L3-NA caused detectable increase in virus-specific serum IgG and local IgA after the third feeding. Immunization with L3-NA increased the survival rate by 34% when the mice were infected using A(H1N1)pdm09 influenza virus after the third feeding. After S. pneumoniae post-influenza infection, the L3-NA-immunized mice were 50% more protected from lethality in comparison with L3-fed mice. Thus, a live probiotic vaccine candidate based on L3 induced the formation of systemic and local immunity and provide partial protection against complicated influenza.

Study of Antibody-Dependent Reactions of Mast Cells In Vitro and in a Model of Severe Influenza Infection in Mice.

We investigated the reaction of mouse peritoneal mast cells (MCs) in vitro after IgG-containing immune complex introduction using A/H5N1 and A/H1N1pdm09 influenza viruses as antigens. The sera of immune mice served as a source of IgG antibodies. The concentration of histamine in the supernatants was determined at 4 hours after incubation with antisera and virus. We compared the contribution of MCs to the pathogenesis of post-immunization influenza infection with A/H5N1 and A/H1N1 influenza viruses in mice. The mice were immunized parenterally with inactivated viruses and challenged with lethal doses of drift A/H5N1 and A/H1N1 influenza viruses on the 14th day after immunization. Simultaneously, half of the mice were injected intraperitoneally with a mixture of histamine receptor blockers (chloropyramine and quamatel). In in vitro experiments, the immune complex formed by A/H5N1 virus and antiserum caused a significant increase in the histamine release compared to immune serum or the virus alone. With regard to the A/H1N1 virus, such an increase was not significant. A/H1N1 immunization caused detectable HI response in mice at 12th day after immunization, in contrast to the A/H5N1 virus. After challenge of A/H5N1-immunized mice, administration of antihistamines increased the survival rate by up to 90%. When infecting the A/H1N1-immunized mice, 90% of the animals were already protected from lethal infection by day 14; the administration of histamine receptor blockers did not increase survival. Histological examination of the lungs has shown that toluidine blue staining allows to estimate the degree of MC degranulation. The possibility of in vitro activation of murine MCs by IgG-containing immune complexes has been shown. In a model of influenza infection, it was shown that the administration of histamine receptor blockers increased survival. When the protection was formed faster due to the earlier production of HI antibodies, the administration of histamine receptor blockers did not significantly affect the course of the infection. These data allow to propose that even if there are antibody-dependent MC reactions, they can be easily stopped by the administration of histamine receptor blockers.

Construction of the Enterococcal Strain Expressing Immunogenic Fragment of SARS-Cov-2 Virus.

Contemporary SARS-Cov-2 pandemic, besides its dramatic global influence on the human race including health care systems, economies, and political decisions, opened a window for the global experiment with human vaccination employing novel injectable vaccines providing predominantly specific IgG response with little knowledge of their impact on the mucosal immunity. However, it is widely accepted that protection against the pathogens at the gates of the infection - on mucosal surfaces-predominantly rely on an IgA response. Some genetically modified bacteria, including probiotics, represent attractive vehicles for oral or nasal mucosal delivery of therapeutic molecules. Probiotic-based vaccines for mucous membranes are easy to produce in large quantities; they have low cost, provide quite a long T-cell memory, and gut IgA response to oral vaccines is highly synchronized and strongly oligoclonal. Here we present a study demonstrating construction of the novel SARS-Cov-2 vaccine candidate employing the gene fragment of S1 SARS-Cov-2 gene. This DNA fragment was inserted in frame into major pili protein gene with d2 domain of enterococcal operon encoding for pili. The DNA sequencing proved the presence of the insert in enterococcal genome. RNA transcription, immunoprecipitation, and immune electron microscopy with human sera obtained from the SARS-Cov-2 patients demonstrated expression of SARS-Cov-2 antigens in bacteria. Taken together the data obtained allowed considering this genetically modified probiotic strain as an interesting candidate for vaccine against SARS-Cov-2.

Mast cell degranulation and histamine release during A/H5N1 influenza infection in influenza-sensitized mice.

Here we evaluate the role of mast cells in infection with influenza A/H5N1 virus in immunized mice. CBA mice were immunized intramuscularly with formalin-inactivated A/Vietnam/1194/2004 (H5N1)NIBRG-14 (H5N1). Serum samples were obtained on days 7, 12, 14, 21 after immunization. At day 14, the mice were infected intranasally with the A/Indonesia/5/2005 (H5N1)IDCDC-RG2 (H5N1) influenza virus with half of the animals receiving a mixture of the antihistamines. 67% of the vaccinated mice were protected from the lethality compared to 43% in the PBS-immunized group. Administration of antihistamines increased survival up to 85%-95%. Immunohistochemical examination using CD117 staining of the lungs demonstrated a larger quantity of activated mast cells after infection of immunized mice compared to mock-immunized mice. This was correlated to increased histamine level in the lungs and blood. Our experimental results suggest the involvement of mast cells and the histamine they produce in the pathogenesis of influenza infection in case of incomplete formation of the immune response to vaccination and mismatch of the vaccine and infection influenza viruses.

Study of Neuraminidase-Inhibiting Antibodies in Clinical Trials of Live Influenza Vaccines.

BACKGROUND: Currently, the immunogenicity of influenza vaccines is assessed by detecting an increase of hemagglutination inhibition (HI) antibodies. As neuraminidase (NA)-based immunity may be significant in protecting against influenza infection, detection of neuraminidase inhibiting (NI) antibodies may improve the assessment of the immunogenicity of influenza vaccines. METHODS: We investigated the immune response to NA in people after immunization with live influenza vaccines (LAIVs). A number of A/H7NX or A/H6NX viruses were used to detect NI antibodies, using an enzyme-linked lectin assay (ELLA). RESULTS: Seasonal LAIV immunization stimulated an increase in NI antibodies not only to homologous A/H1N1 influenza, but also to A/H1N1pdm09 and A/H5N1 influenza. After A/17/California/09/38 (H1N1) pdm09 LAIV vaccination, there was no statistical relationship between post-vaccinated antibody seroconversion and two surface glycoproteins in serum samples obtained from the same individuals (p = 0.24). Vaccination with LAIV of H5N2, H2N2, H7N3, and H7N9 subtypes led to 7%-29.6% NI antibody seroconversions in the absence of HI antibody conversions. There was relatively low coordination of hemagglutinin (HA) and NA antibody responses (r = 0.24-0.59). CONCLUSIONS: The previously noted autonomy for HI and NI immune responses was confirmed when assessing the immunogenicity of LAIVs. Combining the traditional HI test with the detection of NI antibodies can provide a more complete assessment of LAIV immunogenicity.

Mucosal vaccine based on attenuated influenza virus and the group B Streptococcus recombinant peptides protected mice from influenza and S. pneumoniae infections.

Although many influenza-related deaths are attributable to secondary bacterial infection with S. pneumoniae, vaccines that simultaneously protect against influenza and pneumococcal infection are currently not developed. The aim of our study was to evaluate the possibility to prevent post-influenza pneumococcal infection using an associated vaccine based on live influenza vaccine (LAIV) combined with recombinant polypeptides derived from superficial factors of Group B streptococcus (GBS) determining pathogenicity. We demonstrated in a model of post-influenza pneumococcal pneumonia that intranasal pneumococcal super-infection seriously complicated the course of A/Shanghai/2/2013(H7N9) CDC-RG virus infection in mice. Associated immunization using LAIV and GBS vaccine (GBSV) prevented post-influenza pneumococcal pneumonia better than mono-LAIV or GBSV immunization. At the same time, parenteral pneumococcal post-influenza infection of immune mice was more severe in the groups immunized using recombinant GBS peptides which can be explained by antibody-dependent enhancement of infection. In this case, the introduction of blockers of histamine receptors type 1 and 2 reduced the burden of secondary pneumococcal infection.

Factors of early protective action of live influenza vaccine combined with recombinant bacterial polypeptides against homologous and heterologous influenza infection.

We are developing an associated vaccine based on live influenza vaccine (LAIV) and streptococcal recombinant peptides. The recombinant group B streptococcus (GBS) peptides P6 and ScaAB demonstrated a distinguished immunomodulating effect in THP-1 cells. The increase in IFN 1-alpha expression after ScaAB inoculation was similar to that against LAIV. We immunized mice intranasal using of A/H7N3 LAIV or/and ScaAB peptide. At day 5 after immunization, we detected serum IgM which reacted with non-vaccine influenza viruses. Associated vaccination of mice using LAIV and GBS peptide was the most effective against sub-lethal infection with A/H7N9 influenza virus and against lethal challenge with A/H1N1pdm virus at day 5 after immunization. Not only LAIV but also the ScaAB protected about 20% of the immunized animals against lethal challenge with A/H1N1pdm virus. The early protection was related to increasing type 1 interferons expression in the lungs. Our results in mice have shown that successful protection against homologous and heterologous influenza infections can be achieved soon after vaccination with either LAIV or LAIV in combination with GBS recombinant peptide. Presumably, such protection may be mediated by non-specific IgM antibodies and an increase in the expression of early cytokines in the airway.

Anti-neuraminidase antibodies against pandemic A/H1N1 influenza viruses in healthy and influenza-infected individuals.

The main objective of the study was to evaluate neuraminidase inhibiting (NI) antibodies against A/H1N1pdm09 influenza viruses in the community as a whole and after infection. We evaluated NI serum antibodies against A/California/07/09(H1N1)pdm and A/South Africa/3626/2013(H1N1)pdm in 134 blood donors of different ages using enzyme-linked lectin assay and in 15 paired sera from convalescents with laboratory confirmed influenza. The neuraminidase (NA) proteins of both A/H1N1pdm09 viruses had minimal genetic divergence, but demonstrated different enzymatic and antigenic properties. 5.2% of individuals had NI antibody titers ≥1:20 against A/South Africa/3626/2013(H1N1)pdm compared to 53% of those who were positive to A/California/07/2009(H1N1)pdm NA. 2% of individuals had detectable NI titers against A/South Africa/3626/13(H1N1)pdm and 47.3% were positive to A/California/07/2009(H1N1)pdm NA among participants negative to hemagglutinin (HA) of A/H1N1pdm09 but positive to seasonal A/H1N1. The lowest NI antibody levels to both A/H1N1pdm09 viruses were detected in individuals born between 1956 and 1968. Our data suggest that NI antibodies against A/South Africa/3626/13 (H1N1)pdm found in the blood donors could have resulted from direct infection with a new antigenic A/H1N1pdm09 variant rather than from cross-reaction as a result of contact with previously circulating seasonal A/H1N1 variants. The immune responses against HA and NA were formed simultaneously right after natural infection with A/H1N1pdm09. NI antibodies correlated with virus-neutralizing antibodies when acquired shortly after influenza infection. A group of middle-aged patients with the lowest level of anti-NA antibodies against A/California/07/2009 (H1N1)pdm was identified, indicating the highest-priority vaccination against A/H1N1pdm09 viruses.

Prevention of Influenza A(H7N9) and Bacterial Infections in Mice Using Intranasal Immunization With Live Influenza Vaccine and the Group B Streptococcus Recombinant Polypeptides.

We investigate the protective effect of combined vaccination based on live attenuated influenza vaccine (LAIV) and group B streptococcus (GBS) recombinant polypeptides against potential pandemic H7N9 influenza infection followed by GBS burden. Mice were intranasally immunized using 107 50% egg infectious dose (EID50) of H7N3 LAIV, the mix of the 4 GBS peptides (group B streptococcus vaccine [GBSV]), or combined LAIV + GBSV vaccine. The LAIV raised serum hemagglutination-inhibition antibodies against H7N9 in higher titers than against H7N3. Combined vaccination provided advantageous protection against infections with A/Shanghai/2/2013(H7N9)CDC-RG influenza and serotype II GBS. Combined vaccine significantly improved bacterial clearance from the lungs after infection compared with other vaccine groups. The smallest lung lesions due to combined LAIV + GBSV vaccination were associated with a prevalence of lung interferon-γ messenger RNA expression. Thus, combined viral and bacterial intranasal immunization using H7N3 LAIV and recombinant bacterial polypeptides induced balanced adaptive immune response, providing protection against potential pandemic influenza H7N9 and bacterial complications.

Evaluation in Mouse Model of Combined Virus-bacterial Vaccine Based on Attenuated Influenza A(H7N3) Virus and the Group B Streptococcus Recombinant Polypeptides.

BACKGROUND: Secondary bacterial influenza complications are a common cause of excesses morbidity and mortality, which determines the need to develop means for specific prophylaxis. Group B streptococcal infection is especially common cause of pneumonia among children and the elderly with underlying conditions. Here we investigate in a mouse model the effects of combined intranasal immunization using live attenuated influenza vaccine and recombinant polypeptides based on group B Streptococcus surface proteins. METHODS: Groups of outbred mice received two doses of the following preparations: 1) the reassortant A/17/Mallard/Netherlands/00/95 (H7N3) influenza virus; 2) a mixture of P6, ScaAB, ScpB1 and Stv recombinant GBS proteins (20 µg total); 3) the A(H7N3) influenza vaccine pooled with the four bacterial peptide preparation; 4) control animals were treated with PBS. RESULTS: Intranasal vaccination using LAIV in combination with GBS polypeptides provided advantageous protection against infections with homologous A/Mallard/Netherlands/12/00 (H7N3) wild type virus or heterologous A/Puerto Rico/8/34 (H1N1) followed by serotype II GBS infection. Also, combined vaccination improved bacterial clearance from the lungs of mice. CONCLUSION: Intranasal immunization with LAIV+GBSV was safe and enabled to induce the antibody response to each of vaccine components. Thus, the combined vaccine increased the protective effect against influenza and its bacterial complications in mice compared to LAIV-only.

Serum strain-specific or cross-reactive neuraminidase inhibiting antibodies against pandemic А/California/07/2009(H1N1) influenza in healthy volunteers.

BACKGROUND: Pre-existing antibodies to influenza virus neuraminidase may provide protection against infection influenza viruses containing novel hemagglutinin (HA). The aim of our study was to evaluate serum neuraminidase-inhibiting (NI) antibodies against А/California/07/2009(H1N1) [H1N1/2009pdm] and А/New Caledonia/20/1999(H1N1) [H1N1/1999] influenza viruses in relation with the age of participants and hemagglutination-inhibition (HI) antibody levels. Anti-H1N1/2009pdm neuraminidase and anti-H1N1/1999 neuraminidase antibody levels were measured in total 219 serum samples from Russian healthy peoples of various ages examined before and a year after pandemic strain appearance. We adjusted peroxidase-linked lectin micro-procedure to measure NI antibody titers using the reassortant A/H7N1 influenza viruses based on A/equine/Prague/1/56(H7N7). Also, HI antibody titers were estimated against H1N1/2009pdm, H1N1/1999 and a panel of seasonal A/H1N1 influenza viruses. RESULTS: In sera samples collected during the fall of 2010, mean titers of specific HI and NI antibodies to H1N1/2009pdm were 2-2.1 times lower than antibody levels against H1N1/1999. Of the 163 individuals examined, 58 (35.6%) had NI anti-H1N1/2009pdm antibody titers > 1:20, compared to 93 (57.1%) who had NI anti-H1N1/1999 antibody titers > 1:20. There were low correlations between HI and NI antibody levels against either H1N1/1999 or H1N1/2009pdm in the same serum samples. The 24 adults born between 1957 and 1977 expressed very low levels of NI antibodies to A/H1N1 influenza viruses. Persons with low HI anti-H1N1/2009pdm titers but positive to seasonal A/H1N1 demonstrated significantly higher NI anti-A/H1N1 antibody titers than unexposed subjects. In 2005 cross-reactive NI anti-H1N1/2009pdm antibody titers > 1:20 were detected among 7.1% of young people. CONCLUSIONS: Our study confirmed that contact with seasonal influenza viruses may have contributed to generating the cross-reacting anti-H1N1/2009pdm NI antibodies which were detected in the sera of 18-20 years old people examined before the pandemic virus active circulation. The lowest levels of antibodies to the neuraminidase of N1 subtype were in the group of participants born during the circulation of influenza A/H2N2 or A/H3N2 viruses. The low correlation between HI and NI antibody titers suggests that NI antibody detection can be used as an additional test to evaluate the immune response after influenza infections or immunizations.