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NK cells are essential for controlling viral infections. We investigated NK cell and innate lymphoid cell (ILC) dynamics and function in rhesus macaque rectal tissue and blood following mucosal priming with replicating adenovirus (Ad)–SIV recombinants, systemic boosting with SIV envelope protein, and subsequent repeated low-dose intravaginal SIV exposures. Mucosal memory-like NK and ILC subsets in rectal and vaginal tissues of chronically infected macaques were also evaluated. No differences in NK cell or ILC frequencies or cytokine production were seen between vaccinated and Ad-empty/alum controls, suggesting responses were due to the Ad-vector and alum vaccine components. Mucosal NKp44+ ILCs increased postvaccination and returned to prelevels postinfection. The vaccine regimen induced mucosal SIV-specific Ab, which mediated Ab-dependent cellular cytotoxicity and was correlated with mucosal NKp44+CD16+ ILCs. Postvaccination NKp44+ and NKp44+IL-17+ ILC frequencies were associated with delayed SIV acquisition and decreased viremia. In chronically SIV-infected animals, NKp44+ ILCs negatively correlated with viral load, further suggesting a protective effect, whereas, NKG2A− NKp44− double-negative ILCs positively correlated with viral load, indicating a pathogenic role. No such associations of circulating NK cells were seen. Δγ NK cells in mucosal tissues of chronically infected animals exhibited impaired cytokine production compared with non-Δγ NK cells but responded to anti-gp120 Ab and Gag peptides, whereas non-Δγ NK cells did not. Mucosal Δγ NKp44+ and Δγ DN cells were similarly associated with protection and disease progression, respectively. Thus, the data suggest NKp44+ ILCs and Δγ cells contribute to SIV infection outcomes. Vaccines that promote mucosal NKp44+ and suppress double-negative ILCs are likely desirable.

Seasonal vaccination is ineffective in the elderly population and in conferring cross-protection against antigenically different influenza viruses. Therefore, the cross-protective efficacy of influenza vaccines needs to be improved. Here, we compared the effects of different adjuvants (QS-21+MPL, CpG+MPL, BCG CWS, Saponin VSA-1, Quil-A, and Alum) on enhancing the immunogenicity and homologous and cross-protection by influenza vaccination in mice. QS-21+MPL adjuvant was most effective in inducing Th1 T cell, cross-reactive IgG, and hemagglutination inhibiting antibody responses to vaccination. QS-21+MPL and CpG+MPL adjuvants exhibited high potency by preventing weight loss and reducing viral loads and conferring enhanced homologous and cross-protection in young and aged mice. BCG cell-wall skeleton (CWS) displayed lower adjuvant efficacy in inducing Th1 IgG responses and conferring cross-protection. VSA-1 (an analog of licensed adjuvant QS-21) exhibited adjuvant effects on enhancing the protective efficacy against homologous and heterosubtypic viruses comparable to QS-21. Saponin Quil-A exhibited adjuvant effects in CD4-deficient mice suggesting a CD4-independent adjuvant mechanism, in contrast to CpG that requires intact CD4 T cells. The findings suggest that QS-21+MPL, CpG+MPL, and VSA-1 exhibit desirable adjuvant properties on enhancing cross-protective humoral and cellular immunity to vaccination, with a unique pattern of innate immune responses, contributing to improved homologous and heterosubtypic protection. This study has significance in better understanding the effects of potent adjuvants on enhancing homologous and cross-protection to influenza vaccination in young and older adults. This study was supported by NIH/NIAID grants AI093772 (S.M.K.), AI154656 (S.M.K), and AI147042 (S.M.K).

Vaccine adjuvant effects in the CD4-deficient condition largely remain unknown. We investigated the roles of combined monophosphoryl lipid A (MPL) and aluminum hydroxide (Alum) adjuvant (MPL+Alum) in inducing immunity after immunization of CD4 knockout (CD4KO) and wild-type (WT) mice with T-dependent influenza vaccine. MPL+Alum adjuvant mediated IgG isotype-switched Abs, IgG-secreting cell responses, and protection in CD4KO mice, which were comparable to those in WT mice. In contrast, Alum adjuvant effects were dependent on CD4+ T cells. MPL+Alum adjuvant was effective in recruiting monocytes and neutrophils as well as in protecting macrophages from Alum-mediated cell loss at the injection site in CD4KO mice. MPL+Alum appeared to attenuate MPL-induced inflammatory responses in WT mice, likely improving the safety. Additional studies in CD4-depleted WT mice and MHC class II KO mice suggest that MHC class II+ APCs contribute to providing alternative B cell help in the CD4-deficient condition in the context of MPL+Alum–adjuvanted vaccination.

We investigated the adjuvant effects of MPL (TLR4 agonist) and CpG (TLR9 agonist) on promoting protection after vaccination of C57BL/6 and BALB/c mice with inactivated influenza split virus or human respiratory syncytial virus (RSV) vaccines. Adjuvant effects of combination MPL and CpG (MPL+CpG) on improving protective vaccine efficacy were evident by higher levels of protective humoral and cellular immune responses to vaccination, more effective control of lung viral replication, prevention of weight loss, and inflammatory cytokines and cellular infiltrates in vaccinated mice after virus challenge. Vaccine-enhanced respiratory disease, a well-known phenomenon due to atypical immune priming by RSV vaccination after challenge, could be prevented by MPL + CpG adjuvants in RSV vaccination, shaping toward Th1 and IgG2a isotype immune responses. In contrast, aluminum hydroxide and squalene oil-in-water adjuvants (AddaVax) were not effective in conferring vaccine-induced protection against influenza and RSV in BALB/c mice after vaccination and challenge. Combination of MPL and CpG was found to exhibit distinct effects on modulating inflammatory micro-environment with cytokines, chemokines, and innate infiltrates in vivo within a day as well as stimulating dendritic cells in vitro to secrete IL-12p70 and TNF-α cytokines. In summary, the findings suggest that a combination adjuvant of different TLR agonists exhibits a unique pattern of innate and adaptive immune responses, contributing to protection by inactivated influenza split virus and subunit RSV vaccination.

The roles of interferons (IFN) remain poorly understood in generating long-term protective immune responses to vaccination despite their well-known anti-viral innate immunity. We investigated the roles of IFN receptor (IFNR) signaling in inducing adaptive immune responses and protective immunity to influenza virus-like particle (VLP) vaccination by using A129 and AG129 mouse models. AG129 mice showed a significant defect in raising IgG2a Th1 antibody responses despite of the induction of comparable IgG1 Th2 type and hemagglutination inhibiting antibodies compared to those in A129 mice. After lethal challenge infection, the AG129 vaccinated mice were not protected as evidenced by 20% severe weight loss, high airway hyperresponsiveness, histopathology, excessive infiltration of neutrophils, inflammatory chemokine and cytokines, and 80 folds higher lung viral titers compared to the A129 vaccinated mice that were 100% protected without displaying weight loss. AG129 mice were not able to induce rapid recall of plasma cells, germinal center B cells, and IFN-r+ CD4 and CD8 T cells. Depletion of T cells in A129 mice after vaccination resulted in lower efficacy of protection. The role of type I IFNR were determined in AB6 mice by comparing with B6 mice after vaccination. The AB6 mice did not show significant defects in developing IgG1 and IgG2c antibodies and in conferring protection after vaccination and challenge, despite of inability to recruit monocytes, compared to those in B6 mice. These results suggest that type II IFNR signaling plays a more critical role in developing adaptive immunity after vaccination than type I IFNR signaling, and that both type I and II IFNR signaling is required for effective control of influenza virus infection.

Vaccination of young children with alum-adjuvanted formalin-inactivated respiratory syncytial virus (FI-RSV) induced vaccine-enhanced disease (VED) after natural infection during epidemic season, resulting in hospitalizations and two deaths. There is no licensed RSV vaccine. VED was also observed in previous reports with fusion (F) protein RSV vaccines and adjuvants including oil-in-water emulsion, delta-inulin, and natural killer T cell agonist α-GalCer. In this study to search for adjuvants preventing RSV VED, we investigated a novel combination of monophosphoryl lipid A (MPL TLR4 agonist) and oligodeoxynucleotide CpG (CpG TLR9 agonist) in the RSV F protein vaccination. RSV specific IgG antibodies and neutralizing titers, and controlling RSV lung viral loads after challenge were similarly observed in the alum and MPL+CpG adjuvant groups of mice that received a single dose prime immunization with RSV F protein vaccine. Analysis of IgG isotypes and cytokines suggested the induction of T helper type 1 (Th1) immune responses by inclusion of MPL+CpG adjuvant in contrast to alum adjuvant biasing Th2 type immune responses. Most importantly, lung histopathology and infiltration of inflammatory innate immune cells (eosinophils, neutrophils) were not observed after F protein vaccination with MPL+CpG adjuvant whereas severe lung histopathology was induced after alum adjuvanted F protein vaccination and RSV challenge. The results in this study suggest a novel adjuvant approach to improve efficacy and safety of RSV subunit vaccines.

Heat-killed Lactobacillus casei strain lactic acid bacteria DK128 was investigated as potential probiotics to determine its antiviral effects on influenza virus. Intranasal pretreatment of mice with heat-killed DK128 resulted in the recruitment of various innate immune cells into the airways, conferring broad protection against lethal infection with H1, H3, and H5 subtype influenza viruses, preventing body weight loss, and lowering lung viral loads. The protection against lethal infection as a result of killed-DK128 pretreatment was correlated with significantly reduced infiltrates of natural killer cells, neutrophils, and monocytes as well as with increased alveolar macrophage populations. Mice with killed-DK128-mediated protection against H3N2 primary infection effectively developed heterosubtypic adaptive immunity to H1N1 pandemic virus. Further mechanistic studies using various knockout mouse models discovered that B cells but not CD4 and CD8 T cells were absolutely required for heat-killed DK128-mediated protection. Therefore, this study provides evidence that heat-killed DK128 can be developed as beneficial probiotics and that the cross-talk between innate and adaptive immune cells is playing an essential role in conferring influenza antiviral immunity.

Current influenza vaccines including live attenuated influenza vaccine (LAIV) do not provide cross-protection against drift and potential pandemic strains. We hypothesized that supplementing LAIV with a highly conserved protective antigenic target M2 ectodomain (M2e) would confer broad cross-protection by inducing humoral and cellular immune responses to conserved antigenic targets (M2e, NP), overcoming strain-specific protection of current vaccines. Intranasal vaccination with LAIV (A/NL/09/H1N1) supplemented with tandem repeat M2e containing virus-like particles (M2e5x VLPs) induced M2- and H1N1 virus-specific antibodies. Upon heterosubtypic challenge with pandemic potential rgA/H5N1 virus, we found that M2e5x VLP supplemented LAIV immune mice showed significantly improved heterosubtypic protection by preventing weight loss and lowering lung viral titers compared to the LAIV alone group inducing poor heterosubtypic immunity. Further mechanistic studies on heterosubtypic immunity suggest that recall T cell responses to M2e and nucleoprotein as well as systemic and mucosal M2e antibodies were found to be major heterosubtypic immune correlates. Therefore, this study demonstrates a novel vaccination strategy to confer universal protection by supplementing current strain-specific vaccines with a highly conserved cross protective antigenic target M2e5x VLPs.

The action mechanisms of adjuvants have not been well understood yet. We hypothesized that an effective adjuvant would overcome a defect in CD4+ T cell help for the induction of immune responses and protection by T cell dependent vaccines. We found that current influenza split vaccine was not able to induce antibodies and protection in a CD4+ T cell deficient condition. In an attempt to develop an effective adjuvant, we developed a recombinant virus-like particle expressing flagellin (FliC-VLP) and investigated its adjuvant effects and mechanisms by comparing alum adjuvant. CD4 knockout (CD4KO) mice that were immunized with influenza split vaccine plus FliC-VLP (or MF59, AS04 human adjuvant) induced IgG isotype-switched antibodies, germinal center formation, and virus-specific memory cells, all of which were not induced in CD4KO mice with split vaccine alone. Levels of antibodies and protective efficacy in CD4KO mice with split vaccine plus FliC-VLP were significantly higher than those in C57BL/6 wild type mice without adjuvant and CD4KO mice adjuvanted by alum. The adjuvanticity of FliC-VLP was partially dependent on TLR5 when tested in TLR5KO mice. Taken together, the results provide evidence that FliC-VLP adjuvant can stimulate the induction of antigen-specific immune responses and improve protection against lethal infection in CD4KO mice immunized with T cell dependent split vaccines, indicating CD4+ T cell independent action mechanisms of adjuvant.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection and deaths in infants. Since the failure of formalin-inactivated RSV (FI-RSV) due to vaccine-enhanced pulmonary eosinophilia and pneumonia during 1960s, there is no licensed vaccine against RSV. In this study, we analyzed immune cell phenotypes and cytokines in bronchoalveolar lavage fluids (BALF) from mice immunized with FI-RSV, live RSV, or F plasmid and virus-like particles (VLP) containing RSV F and G glycoproteins (FFG VLP) at day 5 post RSV challenge. FI-RSV immunized mice showed severe eosinophilia, high levels of CD11b+ cells as well as IFN-gamma and IL-4 secreting CD4 T cells locally and systemically. Mice pre-immunized with live RSV showed no severe pulmonary eosinophilia, similar levels of IFN-gamma secreting CD4 and CD8 T cells, and low levels of IL-4 secreting cells. Mice immunized with FFG VLPs displayed no eosinophilia, relatively a low ratio of CD4/CD8 T cells secreting IFN-gamma, and no systemic cytokines. All 3 types of RSV vaccines were effective in clearing lung viral loads. Taken together, these results suggest no correlation between lung RSV clearance and inflammatory pulmonary disease, and that FFG VLP can be a promising RSV vaccine candidate conferring protection without lung disease.