A neonatal oral Mycobacterium tuberculosis-SIV prime / intramuscular MVA-SIV boost combination vaccine induces both SIV and Mtb-specific immune responses in infant macaques

Abstract

Mother-to-child-transmission of HIV by breast-feeding remains a major obstacle in the eradication of HIV infection. Compared to adults, HIV-infected infants have more rapid disease and show higher susceptibility to co-infections like tuberculosis (TB). Although the Bacille Calmette-Guérin vaccine can be administered at birth to protect against TB, BCG can disseminate in HIV-infected infants and increase mortality. Thus, a pediatric combination vaccine to stop both HIV and TB infection in infants is urgently needed. Towards the goal of developing a pediatric combination HIV-TB vaccine to prevent both oral HIV acquisition by breast-feeding and TB infection, we tested and optimized an immunization regimen using a novel live attenuated Mycobacterium tuberculosis vaccine engineered to express simian immunodeficiency (SIV) antigens followed by heterologous MVA-SIV boosting in the infant macaque model. A single oral dose of the attenuated Mtb-SIV vaccine strain mc²6435 during the first week of life was sufficient to induce persistent TB-specific immune responses. SIV-specific immunity was induced at low but comparable magnitudes after oral or intradermal priming, and was enhanced following MVA-SIV boosts. T cell responses were most pronounced in intestinal tissues and oral lymph nodes. Importantly, in addition to plasma SIV-specific IgG and IgA antibodies, infant macaques developed mucosal SIV-specific IgA in saliva and intestinal IgA and IgG. While future SIV and Mtb challenge studies will be needed to determine the protective efficacy of the Mtb-SIV / MVA-SIV vaccine, infants at high risk for oral HIV acquisition by breast-feeding and TB infection could profoundly benefit from an effective combination vaccine.

Figure 1. Mtb-specific T cell responses in PBMC

Panel A and Panel B: CD4⁺ and CD8⁺ T cytokine responses after in vitro BCG or PPD stimulation of longitudinally collected blood samples from a representative animal vaccinated with strain mc²6020 (Panel B) or with strain mc²6435 (Panel C), respectively. Note, only single-functional responses are shown. Panel C: Polyfunctional T cell responses in a representative animal in longitudinally collected blood samples. Panel D: T cell responses are induced against a number of Mtb-specific antigens (PBMC data from a representative animal). Panel E: The percentage of animals with vaccine-induced single and polyfunctional PPD-specific T cell responses in tissues. T cells were isolated from selected tissues (tonsil, submandibular LN, ileum, colon and axillary LN) from all animals in Group B. Using a gray scale gradient, the percentage of vaccinated animals that have PPD-specific cytokine producing T cells in all 5 tissues (indicated as ‘5’) is indicated in black, whereas animals lacking PPD-specific responses in all 5 tissues (indicated as ‘0’) are shown in white. Data are stratified by CD4⁺ and CD8^* T cells. CFP, Mtb culture filtrate protein; lysate, Mtb whole cell lysate; BCG, bacille Calmette-Guérin vaccine strain; Ag85b, antigen 85b.

Figure 2. SIV-specific peripheral blood T cell responses

Panel A: Cytokine production following SIV Gag stimulation in peripheral T cells at week 3 for all mc²6435-primed animals in Group B. Panel B: CD4⁺ (left panel) and CD8⁺ (right panel) T cell cytokine production in response to in vitro SIV Gag stimulation in longitudinally collected blood samples of a representative animal after vaccination. Panel C: SIV-specific single, dual and triple positive cytokine responses in peripheral blood CD4⁺ (left panel) and CD8⁺ (right panel) T cells at weeks 3 (post-prime, pre-boost), 8 (post-boosts) and 16. Distinct cytokines or combinations thereof are indicated by different colors in the graph legend.

Figure 3. Homing and SIV-specific T cell responses in tissues

Panel A: Animals primed with strain mc²6435 and boosted with MVA-SIV (gray bars) have higher percentages of memory and effector T cells expressing CD103 compared to unvaccinated animals (white bars). Memory and effector/effector memory cells were defined within the CD4⁺ or CD8⁺ T cell populations as CD45RA⁻CCR7⁻ (T_CM) or CD45RA^+/−CCR7⁻ (T_E/EM), respectively, before measuring CD103 expression. Statistical significance using a one-tailed t-test was calculated using log-transformed values. Shown are mean values ± SEM. Tissues are abbreviated as follows: tonsil (TON), submandibular lymph node (SUB), retropharyngeal lymph node (RET), ileum (ILE), colon (CO), mesenteric lymph node (MES), axillary lymph node (AX), and PBMC. Panel B: CD4⁺ (left panel) and CD8⁺ (right panel) T cell single cytokine production in response to in vitro SIV Gag stimulation of tissue cell populations. Each bar represents the sum of all single cytokine responses in the relevant tissue (see legend for cytokine color coding). Shown are data for all animals that were vaccinated orally with strain mc²6435 and boosted with MVA-SIV. The three bars for each animal represent from left to right: colon (C), ileum (I), and tonsil (T). Panel C: The percentage of animals with vaccine-induced single and polyfunctional T cell responses to SIV Gag (left panel) or SIV Env (right panel). T cells were isolated from tonsil, submandibular LN, retropharyngeal LN, ileum, colon, mesenteric LN, and axillary LN of all vaccinated animals. Data are stratified for single and dual cytokine producing CD4⁺ and CD8^* T cells. Using a gray scale gradient, animals with T cells from all seven tissues producing no cytokine(s) in response to SIV are indicated by ‘0’ (white) and animals with all seven tissues producing cytokine(s) are shown as ‘7’ (black).

Figure 4. SIV-specific plasma and mucosal IgG and IgA antibodies

Panel A and Panel B: Anti-SIV Gag, Pol or anti-SIV Env IgG (left graphs) and IgA (right graphs) antibody development in plasma following oral priming with strain mc²6435 plus MVA-SIV boosts. Note that IgG values are represented on a log scale and IgA values are plotted linearly. Panel C: SIV Gag, Pol (left panel) and Env (right panel) specific activity for IgA in saliva following priming with strain mc²6435 plus MVA-SIV boosts. Panel D: The specific activity for intestinally-secreted anti-SIV Gag, Pol (black) and anti-SIV Env (gray) IgA (left) and IgG (right) antibodies following priming with strain mc²6435 plus MVA-SIV boosts. Intestinal antibodies were only measured at the time of euthanasia. One (*) or two stars (**) above bars indicate that antibody measurements for anti-SIV Env or anti-SIV Gag, Pol, respectively, exceeded mean background levels ± 3SD.