Immunogenicity of recombinant Modified Vaccinia Ankara following a single or multi-dose vaccine regimen in rhesus monkeys

Abstract

Modified Vaccinia Ankara (MVA) is a replication-defective strain of vaccinia virus (VV) that is being investigated in humans as an alternative vaccine against smallpox. Understanding the parameters of a MVA vaccine regimen that can effectively enhance protective immunity will be important for clinical development. The present studies utilize cohorts of rhesus monkeys immunized with recombinant MVA (rMVA) or recombinant VV (rVV) vaccine vectors to investigate the magnitude, breadth, and durability of anti-VV immunity elicited by a single or multi-dose vaccine regimen. These data demonstrate that a single immunization with rMVA elicits weaker cellular and humoral immunity compared to a single inoculation with rVV. However, vaccine-elicited antibody responses, but not T cell responses, are significantly enhanced with repeated immunizations of rMVA. Importantly, only monkeys receiving up to four inoculations with rMVA generated neutralizing antibody (NAb) responses that were comparable in magnitude and durability to those elicited in monkeys receiving two inoculations with rVV. These data also show that the breadth of antibody responses against protein antigens associated with two antigenically distinct forms of infectious VV are similar in rMVA- and rVV-immunized monkeys. Together, these studies suggest that a multi-dose vaccine regimen utilizing up to four inoculations of MVA generates robust and durable antibody-mediated immunity comparable to that elicited by replication-competent VV.

Figure 1

Schematic representation of two vaccine studies in rhesus monkeys utilizing recombinant poxvirus vaccine vectors. rDNA, rVV, rMVA, and rFPV are recombinant vectors expressing SIV Gag and HIV-1 Env vaccine inserts. (A) Monkeys (7 per group) received rDNA prime/recombinant poxvirus or rDNA boost vaccinations as described in Materials and Methods. (B) Monkeys (6 per group) received recombinant poxvirus prime/recombinant poxvirus boost vaccinations. All poxvirus immunizations were administered by intramuscular and intradermal injections with 2×10⁹ pfu.

Figure 2

Anti-VV NAb and cellular immune responses elicited by rVV, rMVA, and rFPV vaccine vectors. Plasma and PBMC samples were obtained from vaccinated monkeys 4 weeks following boost immunizations with either plasmid DNA, rFPV, rMVA or rVV. (A) Serial dilutions of plasma samples were tested for NAb activity against VV:Luc. Data are presented as the mean ID50 neutralization titer from seven monkeys per group +/- SEM. The dashed line represents the assay limit of detection (ID50 titer >20). PBMC were assessed for IFN-γ ELISPOT responses against VV:WR (B) or the vaccine insert, HIV-1 Gag (C). Data are presented as the mean number of antigen-specific SFC per 10⁶ PBMC +/- SEM from seven monkeys per group.

Figure 3

Antibody responses elicited by poxvirus prime/boost immunizations. Plasma samples were obtained from vaccinated monkeys at various timepoints following prime/boost immunizations with rVV, rMVA, and rFPV as indicated. Serial dilutions of samples were tested for antibody binding activity against VV:WR (A) or MVA (B) by ELISA. Data are presented as the mean endpoint titer from six monkeys per group +/- SEM.

Figure 4

Neutralizing antibody responses elicited by poxvirus prime/boost immunizations. Plasma samples were obtained from vaccinated monkeys at various timepoints following prime/boost immunizations with rVV, rMVA, and rFPV as indicated. Serial dilutions of samples were tested for NAb activity against VV:Luc (A) or MVA:Luc (B). The timing of prime and boost immunizations are indicated by arrows. Data are presented as the mean ID₅₀ NAb titer +/- SEM from six monkeys per group.

Figure 5

Antibody responses to vaccinia IMV- and EEV-associated proteins following immunization with either rVV or rMVA. Plasma samples were obtained from vaccinated monkeys at week 13 following priming immunizations with either rVV or rMVA. Serial dilutions of samples were tested by ELISA for antibody binding activity against the IMV-associated proteins L1R and A27L, and the EEV-associated proteins A33R and B5R. Data are presented as the mean endpoint titer +/- SEM from either 6 (rVV prime) or 12 (rMVA prime) monkeys per group.

Figure 6

Protein array analysis of antibody responses to a panel of vaccinia antigens. Plasma samples were obtained from vaccinated monkeys at week 13 following priming immunizations with either rVV or rMVA and tested at a 1:250 dilution against the indicated protein antigens by ELISA. VIG was used as a positive control (20 μg/ml). The baculovirus-produced A27L, A33R, B5R, and L1R recombinant proteins used in the standard ELISA assays described in Figure 5 were included as positive controls, and are shown in the last four rows separated from the main array. Data are presented as response at 4 weeks following subtraction of the response from matched pre-immune plasma. Background responses were consistently below 0.04, a response of 0.05-0.1 was considered borderline and a response above 0.1 as positive. Mean replicate variation was 2.1% +/- 2.3%.

Figure 7

Anti-VV cellular immune responses elicited by poxvirus prime/boost immunizations. PBMC were isolated from vaccinated monkeys at weeks 2 (post-prime 1), 20 (post-prime 2), and 34 (post-boost 1) following immunization. Cellular immune responses against VV:WR were assessed by IFN-γ ELISPOT assays. The data are presented as the mean number of antigen-specific SFC per 10⁶ PBMC +/- SEM from six monkeys per group.