Chimeric Hemagglutinin-Based Influenza Virus Vaccines Induce Protective Stalk-Specific Humoral Immunity and Cellular Responses in Mice

Abstract

The high variation of the influenza virus hemagglutinin (HA), particularly of its immunodominant head epitopes, makes it necessary to reformulate seasonal influenza virus vaccines every year. Novel influenza virus vaccines that redirect the immune response toward conserved epitopes of the HA stalk domain should afford broad and durable protection. Sequential immunization with chimeric HAs (cHAs) that express the same conserved HA stalk and distinct exotic HA heads has been shown to elicit high levels of broadly cross-reactive Abs. In the current mouse immunization studies, we tested this strategy using inactivated split virion cHA influenza virus vaccines (IIV) without adjuvant or adjuvanted with AS01 or AS03 to measure the impact of adjuvant on the Ab response. The vaccines elicited high levels of cross-reactive Abs that showed activity in an Ab-dependent, cell-mediated cytotoxicity reporter assay and were protective in a mouse viral challenge model after serum transfer. In addition, T cell responses to adjuvanted IIV were compared with responses to a cHA-expressing live attenuated influenza virus vaccine (LAIV). A strong but transient induction of Ag-specific T cells was observed in the spleens of mice vaccinated with LAIV. Interestingly, IIV also induced T cells, which were successfully recalled upon viral challenge. Groups that received AS01-adjuvanted IIV or LAIV 4 wk before the challenge showed the lowest level of viral replication (i.e., the highest level of protection). These studies provide evidence that broadly cross-reactive Abs elicited by cHA vaccination demonstrate Fc-mediated activity. In addition, cHA vaccination induced Ag-specific cellular responses that can contribute to protection upon infection.

FIGURE 1. Chimeric HA-based universal influenza virus vaccine concept.

(A) Humans are repeatedly exposed to circulating H1N1 influenza viruses by infection or vaccination. Such repeated exposure induces Abs against the immunodominant HA head domain. (B) By using chimeric influenza Ags sharing the same H1 stalk but different HA head domains (pictured in blue and pink), the immune response can be redirected toward the otherwise immuno-subdominant HA stalk epi-topes. The immunosilencing of the HA head also allows redirecting of the immune response to other immuno-subdominant domains of the virus (including the NA; not shown here).

FIGURE 2. Experimental design for serological analyses.

BALB/c mice were experimentally primed with QIV on day 0, followed by IIV8 on day 28 and IIV5 on day 56 or IIV5 on day 28 and IIV8 on day 56. IIV Ags were adjuvanted with AS01 or AS03 or administered nonadjuvanted. Control groups consisted of two injections of QIV (standard of care) or three injections of PBS. Blood samples were collected on days 0, 28, 56, and 84.

FIGURE 3. Anti-H1 stalk Ab titers.

After priming with QIV, BALB/c mice were immunized with different formulations, as described in Fig. 2, and the levels of elicited anti-H1 stalk IgG were measured by ELISA in sera collected at different time points. A recombinant cH6/1 HA was used as an Ag to measure H1 stalk Abs. (A) Ab titers in pooled sera are shown over time on days 0, 28 (postpriming), 56 (after first immunization), and 84 (after second immunization). (B) Mean Ab titers in individual sera on day 84 are shown. Each symbol represents one mouse. ****p , 0.0001.

FIGURE 4. Cross-reactivity of the anti-H1 Abs.

(A) HA phylogenic relationship between the different evaluated subtypes. Mice were immunized with a vaccine containing the HA stalk domain of H1, and cross-reactivity of the elicited Abs was evaluated in pooled sera to full- length H2 (B), H18 (C), H9 (D), and H3 (F) HAs. The reactivity to H9 HA was also measured in individual mouse sera (E). Bars are means. *p , 0.05, ****p , 0.0001. The levels of N1-reactive Abs are shown in (G).

FIGURE 5. Anti-H1 stalk IgG1 and IgG2a titers.

After priming with QIV, BALB/c mice were immunized with different formulations, as described in Fig. 2, and the levels of elicited anti-H1 IgG1 and IgG2a were measured by ELISA in sera collected on day 84 (after second immunization). To measure H1 stalk Abs, a recombinant cH6/1 HA was used as an Ag for ELISA. In (A) and (C), the levels of IgG1 and IgG2a are shown, respectively, expressed as the absorbance at 490 nm of several 3-fold dilutions of pooled sera. In (B) and (D), the mean IgG1 and IgG2a titers of individual sera are shown. Bars indicate geometric means. Each symbol represents one mouse. (E) Calculated IgG2a/IgG1 ratio for pooled sera. (F) Calculated IgG2a/IgG1 ratio for individual sera. Bars are means. *p , 0.05, **p , 0.01.

FIGURE 6. Functionality of the vaccine-elicited anti-H1 stalk Abs.

After priming with QIV, BALB/c mice were immunized with different formulations, as described in Fig. 2, and the functionality of the elicited anti-H1 stalk Abs collected on day 84 was evaluated in vitro by using an ADCC reporter assay. The specific activation of effector cells against cH6/1N5- (A) or H1N1pdm09 (B)-infected MDCK cells was analyzed by the measurement of luciferase activity. Sera collected on day 84 were also pooled and transferred into naive BALB/c mice. These mice were then challenged with a lethal dose of cH6/1N5 virus (C and D) or H1N1pdm09 virus (E and F), and their weight (C and E) and survival (D and F) were monitored until recovery (16 or 14 d, respectively).

FIGURE 7. Experimental design for cellular immunity testing.

C57BL/6 mice were primed with QIV on day 0, followed by different combinations of vaccines on days 28 and 56, as indicated in Table I. T cell analysis occurred on day 66. On day 84, a subset of animals was challenged with H1N1pdm09, after which the T cell responses were analyzed on day 90. Blood samples for Ab titer determination were collected on days 28, 56, and 84.

FIGURE 8. IFN-γ–producing cells in the spleen and lungs postimmunization.

After priming with QIV, C57BL/6 mice were immunized with different formulations, as described in Fig. 7, and the spleens and lungs of the animals were collected on day 66 to quantify the Ag-specific T cells in these organs. The cells isolated from the spleen (A–C) or the lungs (D–F) were stimulated with either the NP-derived peptide ASNENMETM (NP specific) (A and D), whole virus H1N1pdm09 (B and E), or the irrelevant peptide RAHYNIVTF (derived from human papillomavirus E7) (C and F), and the number of IFN-γ–producing cells was measured by ELISPOT. **p , 0.01, ***p , 0.001, ****p , 0.0001.

FIGURE 9. Direct challenge and viral titers in lungs.

After priming with QIV, C57BL/6 mice were immunized with different formulations, as described in Fig. 7, and the animals were challenged on day 84 with H1N1pdm09 virus. The evolution over time of the anti-H1 stalk Ab titers up to the day of challenge is shown in (A). Weight loss postchallenge was monitored for 6 d (B) up to day 90, and on day 90, lungs were collected, and the viral titers were evaluated by plaque assay (C). Bars are means. **p , 0.01, ****p , 0.0001.

FIGURE 10. IFN-γ–producing cells in the spleen and lungs postchallenge.

After priming with QIV, C57BL/6 mice were immunized with different formulations, as described in Fig. 7, and the animals were challenged on day 84 with H1N1pdm09 virus. Their spleens and lungs were collected on day 90 to quantify the Ag-specific T cells in these organs. The cells isolated from the spleen (A–E) or the lungs (F–I) were stimulated with either the NP-derived peptide ASNENMETM (NP specific) (A and F), a pool of peptides spanning the influenza virus NP (NP overlap) (B), whole virus H1N1pdm09 (C and G), a pool of peptides spanning the H1N1pdm09 HA (HA overlap) (D and H), or the irrelevant peptide RAHYNIVTF (derived from human papillomavirus E7) (E and I), and the number of IFN-γ–producing cells was measured by ELISPOT. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001.