Background:  Cases of exposed but seronegative humans and macaques have previously been reported. An understanding of the mechanisms that account for the control of infection in these exposed seronegative cases should contribute greatly to the development of an effective vaccine.

Methods:  We inoculated 4 to 8 rhesus macaques intravaginally with increasing dose of the pathogenic isolate X4 simian/human immunodeficiency virus (SHIV)_SF33A. Blood samples were collected weekly for assessment of viremia by bDNA, and for CD4⁺ T-cell count and SHIV antibody detection. Surgery was performed at 3 weeks post-inoculation on a subset of animals to obtain tissue samples. T-cell proliferation (Ki67 staining or CFSE) and cytokine production (intracellular staining for interferon-gamma [IFN-g] and interferon-2 [IL-2]) after stimulation of peripheral blood mononuclear cells (PBMC) or tissue cells with viral specific peptides were also assessed.

Results:  Whereas 7 of 8 macaques exposed to >500 TCID₅₀ of X4 SHIV_SF33A were systemically infected, only 1 of 14 macaques exposed to doses ≤500 TCID₅₀ was infected (p = 0.0004). Occult infection, as defined by absence or transient viremia with no evidence of seroconversion but measurable virus-specific T-cell responses in the periphery, was detected in 7 of 8 macaques exposed to a limiting dose (500 TCID₅₀) of the virus. Importantly, in spite of the very weak or undetectable viral load in exposed seronegative macaques, strong T-cell immunity was present in the lymph nodes that drain the genital tract, as well as gut-associated tissue lymph nodes. Upon rechallenge with the same sub-infectious dose 10 weeks after the first, 4 of 7 exposed seronegative macaques became systemically infected. Protection from systemic re-infection was associated with recall of robust antiviral CD8⁺ T-cell immunity, especially at mucosal and lymphoid sites proximal to the site of virus exposure.

Conclusions:  This study led to the identification of the inoculum threshold that is required to establish a generalized infection by vaginal challenge with X4 SHIV_SF33A. Exposure to limiting dose of the virus induced potent localized immune responses which, we propose, may have contributed to limit virus replication following the first encounter, and to subsequently confer protection from systemic infection upon re-exposure.