Highly attenuated vaccine strains of simian immunodeficiency virus protect against vaginal challenge: inverse relationship of degree of protection with level of attenuation

Abstract

Three different deletion mutants of simian immunodeficiency virus (SIV) that vary in their levels of attenuation were tested for the ability to protect against mucosal challenge with pathogenic SIV. Four female rhesus monkeys were vaccinated by intravenous inoculation with SIVmac239Delta3, four with SIVmac239Delta3X, and four with SIVmac239Delta4. These three vaccine strains exhibit increasing levels of attenuation: Delta3 < Delta3X <Delta4. The vaccinated monkeys were challenged by vaginal exposure to uncloned, pathogenic SIVmac251 at 61 weeks after the time of vaccination. On the basis of viral RNA loads in plasma, cell-associated virus loads in peripheral blood, and CD4 cell counts, strong protective effects were observed in all three groups of vaccinated monkeys. However, the degree of protection correlated inversely with the level of attenuation; the least-attenuated strain, SIVmac239Delta3, gave the greatest protection. One monkey in the Delta3X group and two in the Delta4 group clearly became superinfected by the challenge virus, but these animals had levels of SIV RNA in plasma that were considerably lower than those of naive animals that were challenged in parallel. Protection against vaginal challenge appears easier to achieve than protection against intravenous challenge, since four other SIVmac239Delta4-vaccinated monkeys showed no protection when challenged intravenously with a much lower inoculum of the same challenge virus stock. Protection against vaginal challenge in the Delta4-vaccinated group occurred in the absence of detectable serum neutralizing activities and appeared to be associated with the development of an early SIV-specific cytotoxic-T-lymphocyte response. Our results demonstrate that mucosal protection can be achieved by systemic immunization with the highly attenuated SIVmac239Delta4 more than 1 year prior to the time of challenge.

FIG. 1

Cell-associated virus loads in monkeys (Mm) challenged with WT SIVmac251 by the vaginal route. The numbers of infectious cells in PBMC were quantitated as described in Materials and Methods. Code for PBMC load: 0, virus was not recovered even when 10⁶ PBMC were used; 1, virus was recovered with an average of 10⁶ but not fewer PBMC; 2, 333,333 PBMC; 3, 111,111 PBMC; 4, 37,037 PBMC; 5, 12,345 PBMC; 6, 4,115 PBMC; 7, 1,371 PBMC; 8, 457 PBMC.

FIG. 2

Loads measured as amounts of viral RNA in plasma in monkeys (Mm) challenged with WT SIVmac251 by the vaginal route. The dashed lines indicate threefold sensitivity of the assay, i.e., 300 copy equivalents (Eq) per ml.

FIG. 3

Cell-associated virus loads in Δ4-vaccinated monkeys challenged intravenously. Weeks indicate weeks postvaccination with SIVmac239Δ4. The animals were challenged at week 75 intravenously with SIVmac251. The numbers of infectious cells in PBMC are indicated on the y axis. The code for PBMC load is the same as that in the legend to Fig. 1. L41 is an unvaccinated control challenged in parallel.

FIG. 4

Percentages of CD4⁺ T lymphocytes in monkeys (Mm) challenged with SIVmac251 by the vaginal route. The results are expressed as the percentage of cells in PBMC that were CD4⁺ T lymphocytes.

FIG. 5

Percentages of CD4⁺ T lymphocytes in Δ4-vaccinated monkeys challenged intravenously. The animals were challenged intravenously with SIVmac251 at 75 weeks. L41 is the unvaccinated control challenged in parallel. The results are expressed as the percentage of cells in PBMC that were CD4⁺ T lymphocytes.

FIG. 6

Anamnestic antibody responses in monkeys (Mm) challenged vaginally. Plasma from monkeys obtained at the indicated weeks were reacted with purified, lysed SIV, using 1:200 dilutions of plasma and 1:100 dilutions of conjugate. Week 0 is the time of challenge.

FIG. 7

Antibody titers to SIV on the day of vaginal challenge. Plasma obtained from monkeys on the day of, and just prior to, vaginal challenge were reacted with purified, lysed SIV, using serial fourfold dilutions starting at 1:20.

FIG. 8

Analysis of envelope-specific anti-SIV antibody responses on the day of vaginal challenge. Serum samples were analyzed for reactivity to SIVsmB7 viral envelope glycoproteins in a ConA ELISA as previously described (3). Measurements of conformational dependence were calculated from the ratios of serum antibody reactivities to native versus denatured envelope glycoprotein substrates. Measurements of viral envelope glycoprotein-specific antibody avidity were determined by measuring the resistance of serum antibody-envelope glycoprotein complexes to mild treatment with 8 M urea in the ConA ELISA. Unprotected animals (430-93, 445-93, and 465-93) are represented by open symbols; protected animals are represented by solid symbols.

FIG. 9

Prospective analysis of SIV-specific CTL responses in vaccinated monkeys prior to vaginal challenge. CTL activity following antigen-specific stimulation was examined at multiple E/T ratios, and representative data are shown for an E/T of 40:1 for animals vaccinated with SIVmac239Δ3 (A), SIVmac239Δ3X (B), and SIVmac239Δ4 (C).