An alphavirus-based adjuvant enhances serum and mucosal antibodies, T cells, and protective immunity to influenza virus in neonatal mice

Abstract

Neonatal immune responses to infection and vaccination are biased toward TH2 at the cost of proinflammatory TH1 responses needed to combat intracellular pathogens. However, upon appropriate stimulation, the neonatal immune system can induce adult-like TH1 responses. Here we report that a new class of vaccine adjuvant is especially well suited to enhance early life immunity. The GVI3000 adjuvant is a safe, nonpropagating, truncated derivative of Venezuelan equine encephalitis virus that targets dendritic cells (DCs) in the draining lymph node (DLN) and produces intracellular viral RNA without propagating to other cells. RNA synthesis strongly activates the innate immune response so that in adult animals, codelivery of soluble protein antigens induces robust humoral, cellular, and mucosal responses. The adjuvant properties of GVI3000 were tested in a neonatal BALB/c mouse model using inactivated influenza virus (iFlu). After a single immunization, mice immunized with iFlu with the GVI3000 adjuvant (GVI3000-adjuvanted iFlu) had significantly higher and sustained influenza virus-specific IgG antibodies, mainly IgG2a (TH1), compared to the mice immunized with antigen only. GVI3000 significantly increased antigen-specific CD4(+) and CD8(+) T cells, primed mucosal immune responses, and enhanced protection from lethal challenge. As seen in adult mice, the GVI3000 adjuvant increased the DC population in the DLNs, caused activation and maturation of DCs, and induced proinflammatory cytokines and chemokines in the DLNs soon after immunization, including gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), granulocyte colony-stimulating factor (G-CSF), and interleukin 6 (IL-6). In summary, the GVI3000 adjuvant induced an adult-like adjuvant effect with an influenza vaccine and has the potential to improve the immunogenicity and protective efficacy of new and existing neonatal vaccines.

Importance: The suboptimal immune responses in early life constitute a significant challenge for vaccine design. Here we report that a new class of adjuvant is safe and effective for early life immunization and demonstrate its ability to significantly improve the protective efficacy of an inactivated influenza virus vaccine in a neonatal mouse model. The GVI3000 adjuvant delivers a truncated, self-replicating viral RNA into dendritic cells in the draining lymph node. Intracellular RNA replication activates a strong innate immune response that significantly enhances adaptive antibody and cellular immune responses to codelivered antigens. A significant increase in protection results from a single immunization. Importantly, this adjuvant also primed a mucosal IgA response, which is likely to be critical for protection during many early life infections.

FIG 1

GVI3000 is an effective adjuvant to iFlu after a single dose in mice immunized as neonates. Three to five 7-day-old BALB/c mice per experimental group were primed through footpad (f.p.) injection with iFlu (2 μg) with 10⁵ IU of GVI3000 (black bars) or without GVI3000 (white bars). (A to C) Three, 6, 9, and 15 weeks after the immunization, influenza virus-specific IgG (A) and IgG1 (B) and IgG2a (C) antibodies were measured in serum by ELISA. (D) GVI3000 induces mucosal immune response in neonatally primed mice. Five or six 7-day-old BALB/c mice were primed through f.p. injection with iFlu (1 μg) only or with 10⁵ IU of GVI3000. Ten weeks after the mice were primed, all groups were boosted with iFlu (1 μg) alone. Ten days after the boost, antigen-specific IgA were measured in fecal extracts by ELISA. The amount of IgA produced is presented as nanograms of influenza virus-specific IgA per microgram of total IgA. (E) Antibodies with influenza virus hemagglutination inhibition activity were measured by hemagglutination inhibition assay (HAI) at 6 weeks after the immunization. All data are presented as means plus standard errors of the means (SEM) (error bars). Values that are significantly different from the value for iFlu alone by the Mann-Whitney test are indicated by bars and asterisks as follows: *, P < 0.05; **, P < 0.01.

FIG 2

GVI3000 adjuvant promotes influenza virus-specific cellular immune response in DLNs from neonatal mice after a single neonatal immunization. Three 7-day-old BALB/c mice were primed in both rear footpads (f.p.) with iFlu (2 μg) in the presence or absence of 10⁵ IU of GVI3000. As a control adjuvant, three mice from one litter were immunized with alum (1:2 by volume). Eight days after immunization, both popliteal draining lymph nodes from each mouse were harvested and pooled. DLN cells from each experimental group were stimulated with immunodominant CD4 and CD8 HA peptides followed by IFN-γ release ELISPOT assay. Data were normalized to the mock-stimulated positive spots for each experimental group and graphed as the number of IFN-γ-positive spots per 10⁶ DLN cells. Data are presented as means plus SEM. Values that are significantly different (P < 0.05) from the value for iFlu alone by the Mann-Whitney test are indicated by a bar and asterisk.

FIG 3

GVI3000-adjuvanted iFlu protects neonatally immunized mice from a lethal influenza virus challenge. Neonatal mice were immunized with iFlu only (n = 5) or with either GVI3000 (10⁵ IU) (n = 5) or alum (1:5) (n = 7). Included in the challenge were PBS-immunized mice (n = 8). All mice were intranasally challenged with a lethal dose of 10⁶ EID₅₀ of influenza A/PR/8/34 virus at 3 weeks after immunization. (A to F) Mice were monitored for mortality (A and B) and weight (C to F) for 7 days after the challenge.

FIG 4

Viral titers in nasal tissues and lungs of challenged mice. Groups of five 7-day-old mice were immunized with either iFlu (1 μg) alone or with an adjuvant: GVI3000 (10⁵ IU) or alum (Alhydrogel [1:5]). An additional PBS-immunized group was included. Five weeks after the single immunization, mice were challenged intranasally with 10⁶ EID₅₀ of influenza A/PR/8/34 virus. (A) Mice were euthanized 48 h postchallenge, and nasal tissues were collected in TRIzol. Following RNA extraction, real-time PCR was done with PR8 HA primer/probes. C_T values were plotted on PR8 HA standard curve to calculate virus titer. (B) Mice were euthanized 3 days postchallenge, and their lungs were harvested. After homogenization of lung tissues, TCID₅₀ assay was performed on MDCK cells. Cell supernatants were collected after 48 h of incubation, and hemagglutination assay was performed with 5% chicken red blood cells. TCID₅₀/ml values were calculated using the Reed-Muench method (50). All data are presented as means plus SEM. Values that are significantly different by the Mann-Whitney test are indicated by bars and asterisks as follows: *, P < 0.05; **, P < 0.01.

FIG 5

GVI3000 enhances inflammation in the DLNs of neonatal mice. Five 7-day-old BALB/c mice were immunized in both f.p. with PBS only or with iFlu (1 μg) in the presence of 10⁵ IU of VEE replicon particles labeled with GFP (VRP-GFP) or absence of VRP-GFP. At 12 hpi, both popliteal DLNs from each neonatal mouse were harvested, combined, and manually disrupted into a single-cell suspension. (A) DLN cellularity was assessed by using the volume analyzed by an Accuri C6 flow cytometer (BD) and back-calculating the starting sample amount. (B) Representative histogram for inflammatory dendritic cell gating. (C) Using fluorescent antibody staining, immune cells were stained for DCs (CD11c⁺), inflammatory DCs (CD11c⁺ Ly6C^hi), neutrophils (Ly6G⁺), and macrophages (CD11c⁻ CD11b⁺). The total number of each immune cell present in the DLN sample is shown in the graph. Neut, neutrophils; Macs, macrophages. (D) Cells that were positive for GFP expression were then gated for the surface markers of different immune cells. In panels A and C, the data are presented as means plus SEM. Values that are significantly different by the Mann-Whitney test are indicated by bars and asterisks as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001. Values that are not significantly different (ns) are indicated.

FIG 6

Dendritic cells are activated in VRP-GFP-adjuvanted mice. Three groups of four mice each were immunized with PBS, iFlu (2 μg) only, or iFlu (2 μg) with VRP-GFP (10⁵ IU). Immune cells from the DLNs (two per mice per pool) were harvested and stained at 18 hpi for inflammatory DCs (CD11c⁺ Ly6C^hi). The VRP-GFP-adjuvanted group had both GFP-positive (GFP +ve) and GFP-negative (GFP -ve) DCs. (A to D) Inflammatory DCs were gated for MHCII (A), CD40 (B), CD80 (C), and CD86 (D) surface markers. Data are presented as the mean fluorescence intensity (MFI) ± SEM. Values that are significantly different by the Mann-Whitney test are indicated by bars and asterisks as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 7

GVI3000 induces systemic type I interferon immune response in neonatal mice. At least four 7-day-old BALB/c mice were immunized by injecting the f.p. with Ag (iFlu; 1 μg) (A) or Aĝ (Fluzone; 1 μg) (B) alone or with 10⁵ IU of GVI3000. At 3, 6, 12, 24, and 36 hpi, mice were terminally bled. Type I IFN response was measured by IFN bioassay. Data are presented as the mean plus SEM. Values that are significantly different by the Mann-Whitney test are indicated by bars and asterisks as follows: ***, P < 0.001.