Development Of Vaccines For Genital Herpes Simplex Infection

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Abstract

Herpes simplex virus type 2 (HSV-2) is responsible for most cases of genital herpes and can cause severe disease in the newborn and in immunocompromised persons. We have previously tested the ability of HSV-2 glycoprotein vaccines to prevent HSV-2 infection in seronegative persons or to reduce reactivation of the virus in HSV-2 infected persons who have frequent recurrences of genital herpes. Unfortunately, these vaccine studies were unsuccessful in phase III clinical trials. While these vaccines produced high titers of HSV-2 neutralizing antibodies in the serum, they did not result in protection from primary HSV-2 infection or reactivation. These results suggest that other arms of the immune system, besides induction of antibody, must be important in preventing infection. Therefore, we initiated a series of studies to evaluate the role of cellular immunity, particularly T lymphocytes, in controlling reactivation of HSV-2.[unreadable] [unreadable] HSV-2 establishes a latent infection in neurons of the dorsal root ganglia along the spine, or in neurons in the trigeminal ganglia that innervates the face. Corneal inoculation of mice with HSV-2 results in a latent infection in the trigeminal ganglia. Recent studies have shown that CD8+T lymphocytes in mouse trigeminal ganglia have an important role in suppressing reactivation of HSV from latency. In addition, the number of latent HSV-2 genome copies is also a critical determinant of the rate of reactivation of the virus. Therefore, the balance of between the number of latent viral genomes and the number of CD8+T lymphocytes in the trigeminal ganglia should regulate the rate of reactivation.[unreadable] [unreadable] We postulated that the number of latent viral genomes in trigeminal ganglia is the primary determinant of the rate of virus reactivation, but that differences in the CD8+T lymphocyte responses in the ganglia regulate the reactivation rate. We inoculated mice with different titers of HSV-2 and when the animals were latently infected, they were sacrificed and their trigeminal ganglia were removed. The ganglia were treated with collagenase and a cell suspension was obtained containing both HSV-2 latently infected neurons and CD8+ T lymphocytes. One aliquot of the cell suspension was used to determine the amount of latent virus genomes in the neurons and to quantify the number of CD8+ T lymphocytes in the suspension. Another fraction of the cell suspension was used in an experiment in which the CD8+T lymphocytes were removed (CD8+ T cell depleted cultures) and the ability of the virus to reactivate from the neurons was measured by the detecting infectious HSV-2 released from neurons into the culture. Another aliquot of the cell suspension was used in an experiment in which the CD8+T lymphocytes were kept along with the virus-infected neurons (non-depleted cultures) and the ability of the virus to reactivate from neurons was assayed. The reactivation rates in both CD8+ T lymphocyte depleted and non-depleted cultures significantly correlated with the number of latent HSV-2 genomes, but the correlation was much stronger in CD8+ T lymphocyte depleted cultures. This indicates that CD8+ T lymphocytes have an important role in regulating the rate of HSV-2 reactivation. [unreadable] [unreadable] In another experiment, CD8+ T lymphocytes were isolated from latently infected mouse trigeminal ganglia, and then added back to the CD8+ T cell depleted cultures in increasing numbers. A dose response was observed such that increasing numbers of CD8+ T lymphocytes resulted in a progressive reduction in the rate of reactivation. At a certain threshold of CD8+ T lymphocytes, reactivation was completely prevented. This again confirms the importance of CD8+ T lymphocytes in controlling virus reactivation. [unreadable] [unreadable] Using the data in the above experiments we developed an equation relating the rate of reactivation from neurons with both the number of latent viral genome copies in the trigeminal ganglia and the number of CD8+ T lymphocytes in the ganglia. We then performed a new series of experiments to see if the equation could accurately predict the rate of reactivation of the virus from infected neurons. Mice were infected with three different doses of HSV-2 and when they had become latently infected, the trigeminal ganglia were removed and the number of CD8+ T lymphocytes, latently infected HSV-2 genome copies, and rate of reactivation were determined. We found that the predicted rate of reactivation from the equation correlated significantly with the observed reactivation rate in the cultures. Thus, reactivation of HSV-2 from ganglia in culture is determined by a mathematical relationship based on both the number of latent HSV-2 genome copies in neurons and the number of CD8+ T lymphocytes in the ganglia.