Heterologous prime/boost immunization of rhesus monkeys by using diverse poxvirus vectors

Abstract

As the diversity of potential immunogens increases within certain classes of vectors, the possibility has arisen of employing heterologous prime/boost immunizations using diverse members of the same family of vectors. The present study was initiated to explore the use of divergent pox vectors in a prime/boost regimen to elicit high-frequency cellular immune responses to human immunodeficiency virus type 1 envelope and simian immunodeficiency virus gag in rhesus monkeys. We demonstrated that monkeys vaccinated with a recombinant modified vaccinia virus Ankara (rMVA) prime/recombinant fowlpox virus (rFPV) boost regimen and monkeys vaccinated with a recombinant vaccinia virus prime/rFPV boost regimen developed comparable cellular immune responses that were greater in magnitude than those elicited by a homologous prime/boost with rMVA. Nevertheless, comparable magnitude recall cellular immune responses were observed in monkeys vaccinated with heterologous and homologous recombinant poxvirus following challenge with the CXCR4-tropic SHIV-89.6P. Consistent with this finding, comparable levels of containment of viral replication and CD4(+) T-lymphocyte preservation were seen in these groups of recombinant poxvirus-vaccinated monkeys. This study supports further exploration of combining recombinant vectors of the same family in prime/boost immunization strategies to optimize vaccine-elicited cellular immune responses.

FIG. 1.

Vaccine-elicited cellular immune responses to SIVmac251 Gag and HIV-1 89.6P Env. PBMCs were isolated at weeks 10 (panel 1, postprime), 28 (panel 2, postboost 1), 45 (panel 3, postboost 2), and 64 (panel 4, day of challenge) postimmunization and assessed in IFN-γ ELISPOT assays following in vitro exposure to peptide pools spanning SIV Gag and HIV-1 89.6P Env proteins. The bars represent the means ± standard error of the means (error bars) of SFC responses to individual viral proteins for six monkeys in each group. PBMCs of the control-vaccinated monkeys demonstrated SFC responses of less than 20/10⁶ PBMC.

FIG. 2.

(A) Cellular immunity elicited by recombinant pox vector prime-recombinant pox vector boost immunizations is mediated by both CD4⁺ and CD8⁺ T lymphocytes. IFN-γ ELISPOT responses were measured in freshly isolated unfractionated (top panel) as well as CD8⁺ T-lymphocyte-depleted (bottom panel) PBMCs of the monkeys 2 weeks following the first recombinant pox vector boost immunizations. (B) IFN-γ secretion by CD4⁺ and CD8⁺ T lymphocytes was measured by intracellular cytokine staining following stimulation of PBMCs with peptide pools spanning the HIV-1 Env protein 1 week following the second recombinant pox vector boost. Error bars indicate standard errors of the means.

FIG. 3.

PBMC IFN-γ ELISPOT responses in the vaccinated and control monkeys following viral challenge. Freshly isolated PBMCs were assessed for IFN-γ ELISPOT responses after in vitro exposure to peptide pools spanning the SIVmac251 Gag and HIV-1 89.6P Env proteins at weeks 2 (Wk 2 PC), 4 (Wk 4 PC), and 80 (Wk 80 PC) following intravenous SHIV-89.6P challenge. The bars represent the means ± standard errors of the means (error bars) of values of SFC responses to individual viral proteins for six monkeys in each group of experimentally vaccinated as well as control-vaccinated animals. PC, postchallenge.

FIG. 4.

Plasma viral RNA levels following intravenous challenge with SHIV-89.6P. These values were determined by an ultrasensitive branched DNA amplification assay with a detection limit of 125 copies/ml. Viral RNA levels are shown as the log copies of plasma viral RNA/ml of plasma for individual monkeys for each time point. Red lines indicate the median values for each experimental group at each sampling time.

FIG. 5.

Peripheral blood CD4⁺ T lymphocytes following SHIV-89.6P challenge. The values were determined by multiplying percentage of CD3⁺ CD4⁺ T lymphocytes by the total lymphocyte counts. Red lines indicate the median CD4⁺ T-cell count for each experimental group at each sampling time.

FIG. 6.

Mortality of the SHIV-89.6P challenged monkeys that received experimental or sham vaccines. The P value calculated by the Wilcoxon rank sum test was <0.001.