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. 2017 Sep 12;91(19):e01050-17.
doi: 10.1128/JVI.01050-17. Print 2017 Oct 1.

Synthetic Toll-Like Receptor 4 (TLR4) and TLR7 Ligands Work Additively via MyD88 To Induce Protective Antiviral Immunity in Mice

Peter H Goff  1   2 , Tomoko Hayashi  3 , Wenqian He  1   2 , Shiyin Yao  3 , Howard B Cottam  3 , Gene S Tan  1 , Brian Crain  3 , Florian Krammer  1 , Karen Messer  3 , Minya Pu  3 , Dennis A Carson  3 , Peter Palese  4   5 , Maripat Corr  6
Affiliations

Synthetic Toll-Like Receptor 4 (TLR4) and TLR7 Ligands Work Additively via MyD88 To Induce Protective Antiviral Immunity in Mice

Peter H Goff et al. J Virol. .

Abstract

We previously demonstrated that the combination of synthetic small-molecule Toll-like receptor 4 (TLR4) and TLR7 ligands is a potent adjuvant for recombinant influenza virus hemagglutinin, inducing rapid and sustained immunity that is protective against influenza viruses in homologous, heterologous, and heterosubtypic murine challenge models. Combining the TLR4 and TLR7 ligands balances Th1 and Th2-type immune responses for long-lived cellular and neutralizing humoral immunity against the viral hemagglutinin. Here, we demonstrate that the protective response induced in mice by this combined adjuvant is dependent upon TLR4 and TLR7 signaling via myeloid differentiation primary response gene 88 (MyD88), indicating that the adjuvants function in vivo via their known receptors, with negligible off-target effects, to induce protective immunity. The combined adjuvant acts via MyD88 in both bone marrow-derived and non-bone marrow-derived radioresistant cells to induce hemagglutinin-specific antibodies and protect mice against influenza virus challenge. The protective efficacy generated by immunization with this adjuvant and recombinant hemagglutinin antigen is transferable with serum from immunized mice to recipient mice in a homologous, but not a heterologous, H1N1 viral challenge model. Depletion of CD4+ cells after an established humoral response in immunized mice does not impair protection from a homologous challenge; however, it does significantly impair recovery from a heterologous challenge virus, highlighting an important role for vaccine-induced CD4+ cells in cross-protective vaccine efficacy. The combination of the two TLR agonists allows for significant dose reductions of each component to achieve a level of protection equivalent to that afforded by either single agent at its full dose.IMPORTANCE Development of novel adjuvants is needed to enhance immunogenicity to provide better protection from seasonal influenza virus infection and improve pandemic preparedness. We show here that several dose combinations of synthetic TLR4 and TLR7 ligands are potent adjuvants for recombinant influenza virus hemagglutinin antigen induction of humoral and cellular immunity against viral challenges. The components of the combined adjuvant work additively to enable both antigen and adjuvant dose sparing while retaining efficacy. Understanding an adjuvant's mechanism of action is a critical component for preclinical safety evaluation, and we demonstrate here that a combined TLR4 and TLR7 adjuvant signals via the appropriate receptors and the MyD88 adaptor protein. This novel adjuvant combination contributes to a more broadly protective vaccine while demonstrating an attractive safety profile.

Keywords: TLR4; TLR7; Toll-like receptor; adjuvant; influenza virus; vaccine.

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Figures

FIG 1
FIG 1
1V270/1Z105-induced reduction in lung viral titers correlated with decreased morbidity from influenza virus challenge. WT (BALB/c) mice (4 to 5 animals/group) were immunized with rPR/8 HA adjuvanted with 1V270/1Z105, 1V270, 1Z105, AddaVax, or no adjuvant and subsequently challenged with PR/8 virus 3 weeks after immunization. (A) Virus-challenged mice were monitored for morbidity as measured by weight loss until day 6 postinfection (DPI), when they were sacrificed. Statistical significance of weight loss was assessed by multiple t tests compared to the no-adjuvant group. (B) Lung viral titers from mice sacrificed on day 6 postinfection were quantified by plaque assays. The limit of detection for the plaque assay was 10 PFU/ml. Statistical significance was assessed by an ANOVA for comparison to the no-adjuvant group, and also with the Kruskal-Wallis test. (C) The percentage of body weight lost on day 6 postinfection was plotted against the lung viral titer for each mouse, and a linear regression curve was generated. The Pearson correlation coefficients (r) are shown. *, P < 0.05; **, P < 0.01.
FIG 2
FIG 2
The protective efficacy of the 1V270/1Z105 combined adjuvant is dependent upon TLR4, TLR7, and MyD88. WT (C57BL/6) and Tlr4−/−, Tlr7−/−, Myd88−/−, or Ticam1Lps2 mice were immunized with rPR/8 HA with adjuvant or vehicle alone 3 to 4 weeks before being bled and challenged with PR/8 virus. (A to C) WT (C57BL/6), Tlr7−/−, and Myd88−/− mice (11 to 12 animals/group) were immunized with rPR/8 HA plus 1V270. (A) Total serum IgG was quantified in an ELISA with PR/8 virus substrate, and the mice were subsequently challenged and followed for morbidity (B), based on weight loss, and mortality (C). The statistical significance of weight loss was assessed for each group compared to the WT, as indicated by the colors of the asterisks, by multiple t tests. (D to F) WT (C57BL/6), TLR4−/−, Myd88−/−, and Ticam1Lps2 mice (11 to 17 animals/group) were immunized with rPR/8 HA plus 1Z105. (D) Total serum IgG was quantified by ELISA with PR/8 virus substrate, and the mice were subsequently challenged and followed for morbidity (E), assayed by weight loss, and mortality (F). Statistical significance of weight loss was assessed for each group compared to the WT, as indicated by the colors of the symbols, by multiple t tests. (G to I) WT (C57BL/6), Myd88−/−, and Tlr4.Tlr7−/− mice (6 to 11 animals/group) were immunized with rPR/8 plus 1V270/1Z105, rPR/8 in PBS, or vehicle alone. (G) Total serum IgG was quantified by ELISA with PR/8 virus substrate, and the mice were subsequently challenged and followed for morbidity (H), assayed by weight loss, and mortality (I). Statistical significance of weight loss was assessed for each group compared to the WT by multiple t tests, and only the statistically significant differences between the WT animals immunized with 1V270/1Z105 and the Myd88−/− animals immunized with 1V270/1Z105 are indicated (purple asterisks). (J to L) WT (C57BL/6), Myd88−/−, and Tlr4.Tlr7−/− mice (5 to 6 animals/group) were immunized with rPR/8 HA plus poly(I·C). A second group of WT (C57BL/6) mice (4 animals) was immunized with rPR/8 in PBS. (J) Total serum IgG was quantified by ELISA with PR/8 virus substrate, and the mice were subsequently challenged and followed for morbidity (K), assayed by weight loss, and mortality (L). Statistical significance for IgG endpoint titers, compared to those in WT mice receiving an adjuvanted immunization, was assessed by using the Kruskal-Wallis test. Statistical significance of survival was assessed by the Mantel-Cox test to compare treated and WT mice receiving adjuvanted immunizations as indicated. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
FIG 3
FIG 3
Induction of IgG antibodies and protective efficacy are dependent upon MyD88 expression in bone marrow-derived and radioresistant cells. (A) BM chimeric mice, including Myd88WT BM (n = 23), WTMyd88 BM (n = 21), WTWT BM (n = 24), and Myd88Myd88 BM (n = 10) mice, were immunized with 1V270/1Z105-adjuvanted rPR/8 HA protein at days 0 and 14 and bled on day 35; sera were assayed for total IgG in an ELISA with PR/8 virus substrate. The data shown are pooled from two independent experiments. Bone marrow chimeric mice were challenged with 10 mLD50 of PR/8 virus on day 36 after the first immunization and assayed for morbidity (B), measured by body weight loss, and mortality (C) for each of the following BM chimeric groups: Myd88WT BM (n = 9), WTMyd88 BM (n = 8), WTWT BM (n = 15), and Myd88Myd88 BM (n = 5). Significant differences in body weights compared to the WTWT BM mice are indicated by the asterisk colors. (D) The IgG endpoint titer was plotted against the minimum percentage of initial body weight lost for each animal, and the Pearson correlation coefficient (r) and linear regression line are included. Statistical significance for IgG endpoint titers was assessed by the Kruskal-Wallis test. Statistical significance of weight loss was assessed by t tests adjusted for multiple comparisons. Statistical significance of survival was assessed by the Mantel-Cox test to compare WT mice receiving adjuvanted immunizations, as indicated. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
FIG 4
FIG 4
Passive serum transfer and CD4+ cells induced by 1V270/1Z105-adjuvanted rHA contribute to protection from lethal influenza virus challenge. Immune sera were generated from mice by immunization with rPR/8 or rCal/09 HA adjuvanted with 1V270/1Z105 or no adjuvant at 0 and 3 weeks and administered to recipient mice. Mice immunized with rPR/8 were challenged with homologous PR/8 virus and followed for morbidity (A), as assayed by weight loss, and mortality (B). Mice receiving immune sera from either rPR/8- or rCal/09-vaccinated animals were challenged with NL/09 virus, such that heterologous protection was assayed using homologous protection as the control group, and these animals were followed for morbidity (C), based on body weight loss, and mortality (D). Statistical significance of weight loss was assessed by multiple t tests, comparing the 1V270/1Z015 versus no adjuvant groups (A) and the Cal/09 homologous control (C), as indicated by the colors of the asterisks. (E and F) Mice were immunized with rPR/8 HA plus 1V270/1Z105, and 3 to 4 weeks later they were treated with the MAb GK 1.5 to deplete CD4+ cells or the MAb LTF-2 (isotype) as a control. Two days after administration of the MAbs, mice were challenged with homologous PR/8 virus and assayed for morbidity, based on weight loss, and mortality (animal survival data are shown in parentheses) (E) or with heterologous NL/09 virus and assayed for morbidity by weight loss and mortality (animal survival data are shown in parentheses) (F). For the heterologous challenge, two independent experiments with 10 mice/group and 19 to 20 mice/group were conducted and combined. Statistical significance of weight loss was assessed based on multiple t tests. Statistical significance of survival was assessed with the Mantel-Cox test. *, P < 0.05; **, P < 0.01.
FIG 5
FIG 5
1V270 and 1Z105 work additively to induce antigen-specific IgG and protect mice from morbidity after viral challenge. (A) WT BALB/c mice (4 to 5 animals/group) were immunized with rPR/8 HA with or without adjuvant at the indicated doses or with antigen only in the vehicle control group. Animals were bled 3 weeks after immunization and subsequently challenged with PR/8 virus. Antigen-specific antibody endpoint titers were determined by ELISA with the PR/8 virus substrate, and the endpoint IgG titer for all groups demonstrated a dose response to both single agents 1V270 and 1Z105. The log endpoint titer is a linear function of the log dose of 1V270 (P < 0.05), and 1Z105 has an additive effect on 1V270 (P < 0.0001). However, 1Z105 does not change the 1V270 dose-response relationship, apart from this additive effect (for the overall interaction, P = 0.11), except for the very highest doses (see the text). (B) Body weight data for each group receiving the indicated doses of adjuvant or control were plotted as a function of the endpoint titer. The percent initial body weight change on day 6 following a lethal viral challenge correlated with the endpoint titers (Pearson correlation coefficient r = 0.91, P < 0.0001).
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