key: cord-0842170-hfjggkgi
authors: Nagai, Minami; Moriyama, Miyu; Ichinohe, Takeshi
title: Disruption of nasal bacteria enhances protective immune responses to influenza A virus and SARS-CoV-2 infection in mice
date: 2020-12-26
journal: bioRxiv
DOI: 10.1101/2020.12.25.424300
sha: 8640a6c89d0ddfd231bd3a0c04a3af8c6dc1dfca
doc_id: 842170
cord_uid: hfjggkgi

Gut microbiota plays a critical role in the induction of adaptive immune responses to influenza virus infection. However, the role of nasal bacteria in the induction of the virus-specific adaptive immunity is less clear. Here we demonstrate that while intranasal administration of influenza virus hemagglutinin vaccine alone was insufficient to induce the vaccine-specific antibody responses, disruption of nasal bacteria by lysozyme or addition of culturable oral bacteria from a healthy human volunteer rescued inability of the nasal bacteria to generate antibody responses to intranasally administered the split-virus vaccine. Myd88-depdnent signaling in the hematopoietic compartment was required for adjuvant activity of intranasally administered oral bacteria. In addition, we found that the oral bacteria-combined intranasal vaccine induced protective antibody response to influenza virus and SARS-CoV-2 infection. Our findings here have identified a previously unappreciated role for nasal bacteria in the induction of the virus-specific adaptive immune responses.

Respiratory infectious diseases such as influenza and coronavirus disease 2019 30 (COVID-19) cause substantial morbidity and mortality. Influenza A virus is responsible 31 for annual epidemics that cause severe morbidity and mortality involving 3 to 5 million 32 people worldwide. In addition, the constant pandemic potential of newly emerging 33 end, we immunized mice intranasally with HA and streptococcus salivarius (S. 136 salivarius), streptococcus parasanguinis (S. parasanguinis), or streptococcus infantis (S. 137 infantis). Mice immunized with HA and each isolated bacterial strain induced 138 comparable levels of the HA-specific nasal IgA and serum IgG responses (Fig. 4) , 139

suggesting that adjuvant activity of the oral bacteria is unlikely to account for strain 140 specific. 141 142 Myd88-depdnent signaling in the hematopoietic compartment is required for 143 adjuvant activity of intranasally administered oral bacteria 144

Next, we wished to determine the innate immune signaling through 145 pattern-recognition receptors required for adjuvant activity of the oral bacteria. To this 146 end, we immunized WT and MyD88-deficient mice intranasally with HA and culturable 147 oral bacteria from a healthy volunteer and measured the HA-specific nasal IgA and 148 serum IgG responses. The HA-specific nasal IgA and serum IgG responses were found 149 to be completely dependent on MyD88 (Fig. 5A, B) . In addition, lysozyme-induced 150 disruption of nasal bacteria stimulated the HA-specific nasal IgA and serum IgG 151 responses in a MyD88-dependent manner (Fig. 5C, D) . To determine the cellular 152 compartment responsible for adjuvant activity of oral bacteria, we generated bone 153 11 marrow (BM) chimeric mice in which only the hematopoietic (WT→MyD88 -/-) or the 154 stromal cells (MyD88 -/-→WT) expressed MyD88. After intranasal vaccination with HA 155 and oral bacteria, the HA-specific nasal IgA and serum IgG responses were significantly 156 reduced in MyD88 -/-→WT BM chimeric mice compared to WT→MyD88 -/-BM 157 chimeric mice (Fig. 6) . These data indicate that MyD88-dependent signaling in the 158 hematopoietic, but not stromal, compartment is required for adjuvant activity of 159 intranasally administered oral bacteria. 160 161

Finally, we examined protective effects of intranasal vaccination with oral 164 bacteria-adjuvanted vaccine against influenza virus and SARS-CoV-2 infection. To this 165 end, we immunized mice intranasally with quadrivalent influenza HA vaccine 166 containing A/California/7/2009 HA together with culturable oral bacteria or lysozyme. 167 Two weeks after the second vaccination, we challenged vaccinated mice intranasally 168 with a heterologous A/Narita/1/2009 (pdm09) strain (Fig. 7) . Mice immunized with HA 169 vaccine adjuvanted with oral bacteria or lysozyme significantly reduced the virus titer 170 compared to control mice immunized with the HA vaccine alone (Fig. 7) . We next 12 assessed protective effects of intranasal vaccination with oral bacteria-adjuvanted 172 SARS-CoV-2 spike protein against SARS-CoV-2 infection in Syrian hamsters. To this 173 end, we immunized hamsters intranasally with a recombinant SARS-CoV-2 spike 174 protein and culturable oral bacteria from a healthy volunteer. We immunized hamsters 175 subcutaneously with the spike protein alone as a control. We 176

Both the spike-and the virus-specific serum IgG levels were significantly elevated in 177 immunized hamsters (Fig. 8A, B ). In addition, immunized hamsters significantly 178 reduced the virus titer compared to naïve animals following high-dose (2×10 6 pfu of 179 SARS-CoV-2) challenge (Fig. 8C) . These data collectively indicated that disruption of Oral and nasal washes were collected from a healthy volunteer as described above. 346

Aliquots of 100μl of serial 10-fold dilution of the oral and nasal wash were inoculated 347 into brain heart infusion agar plates (BD 252109). After incubation at 37˚C overnight 348 under the aerobic conditions, the bacterial colonies were grown in brain heart infusion 349 broth (BD 237500) at 37˚C overnight. Bacterial DNA was isolated as described 

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