Acute mucosal pathogenesis of feline immunodeficiency virus is independent of viral dose in vaginally infected cats

Abstract

Background: The mucosal pathogenesis of HIV has been shown to be an important feature of infection and disease progression. HIV-1 infection causes depletion of intestinal lamina propria CD4+ T cells (LPL), therefore, intestinal CD4+ T cell preservation may be a useful correlate of protection in evaluating vaccine candidates. Vaccine studies employing the cat/FIV and macaque/SIV models frequently use high doses of parenterally administered challenge virus to ensure high plasma viremia in control animals. However, it is unclear if loss of mucosal T cells would occur regardless of initial viral inoculum dose. The objective of this study was to determine the acute effect of viral dose on mucosal leukocytes and associated innate and adaptive immune responses.

Results: Cats were vaginally inoculated with a high, middle or low dose of cell-associated and cell-free FIV. PBMC, serum and plasma were assessed every two weeks with tissues assessed eight weeks following infection. We found that irrespective of mucosally administered viral dose, FIV infection was induced in all cats. However, viremia was present in only half of the cats, and viral dose was unrelated to the development of viremia. Importantly, regardless of viral dose, all cats experienced significant losses of intestinal CD4+ LPL and CD8+ intraepithelial lymphocytes (IEL). Innate immune responses by CD56+CD3- NK cells correlated with aviremia and apparent occult infection but did not protect mucosal T cells. CD4+ and CD8+ T cells in viremic cats were more likely to produce cytokines in response to Gag stimulation, whereas aviremic cats T cells tended to produce cytokines in response to Env stimulation. However, while cell-mediated immune responses in aviremic cats may have helped reduce viral replication, they could not be correlated to the levels of viremia. Robust production of anti-FIV antibodies was positively correlated with the magnitude of viremia.

Conclusions: Our results indicate that mucosal immune pathogenesis could be used as a rapid indicator of vaccine success or failure when combined with a physiologically relevant low dose mucosal challenge. We also show that innate immune responses may play an important role in controlling viral replication following acute mucosal infection, which has not been previously identified.

Figure 1

Mean plasma viral RNA. Plasma from blood sampled at weeks 0, 2, 4, 6 and 8 post-infection was evaluated for viral RNA using real-time PCR. Mean viral RNA copies per ml of plasma are shown for high, middle and low dose groups.

Figure 2

Absolute CD4+ T cell count in PBMC. Blood sampled at weeks 0, 2, 4, 6 and 8 post-infection was evaluated for phenotypic expression of CD4+ T cells and calculated based on total WBC counts with differential cell count assessed from cytological evaluation. Mean and standard deviation for absolute CD4+ T cell counts are presented for high, middle and low dose groups (A), and based on presence or absence of viremia (B). Mean and standard errors for the CD4:CD8 ratio are shown for all dose groups combined (C). Statistics were calculated using ANOVA comparing baseline week with post-infection samples within each group. Significance shown using p-values, with * p < 0.01 and # p = 0.06.

Figure 3

Percent CD4+ T cells in peripheral sites eight weeks post-infection. The percent CD4+ T cells is based upon total mononuclear cells isolated. Mean and standard deviation are shown for control, high, middle and low dose groups at euthanasia. No statistically significant differences were identified using ANOVA analysis. PBMC, peripheral blood mononuclear cells; PLN, prescapular lymph node; RLN, retropharyngeal lymph node; ILN, medial iliac lymph node; MLN, mesenteric lymph node; and SPLN, spleen.

Figure 4

Alterations in mucosal lymphocyte populations eight weeks post-infection. Box and whisker plots show the median with upper and lower quartile represented by the boxes, and minimum and maximum values shown by the whiskers. Percent of CD4+ T cells in LPL (A), CD8α+ T cells in IEL (B), and CD8β+ T cells in IEL (C) are shown for control, high, middle and low dose groups at euthanasia. Statistical significance was calculated using ANOVA, with p-value shown for FIV-infected groups compared to controls.

Figure 5

NK cell subsets eight weeks post-infection. Percent NK cells (CD56+) in PBMC, Spleen and IEL for all dose groups of FIV-infected cats versus control cats (A). Percent of NKT cells (B) and classic NK cells (C) are shown based presence or absence of viremia versus control cats. Box and whisker plots show the median with upper and lower quartile represented by the boxes, and minimum and maximum values shown by the whiskers. Statistics were calculated using an unpaired t-test in (A), and based on ANOVA in (B) and (C), with significance shown using p-values where differences were identified.

Figure 6

Intracellular cytokine expression of IFNγ and IL-2 by CD4+ and CD8+ T cells eight weeks post-infection. Expression of IFNγ is shown in the top panels, IL-2 in the middle panels and dual expressing (IFNγ+IL-2+) in the bottom panels for CD4+ T cells (A) and CD8+ T cells (B). Individual data points are shown with mean expression indicated by a line for each data set. Significant differences in cytokine expression are indicated by a line over the applicable data sets and p-value indicated. Calculations were made using an unpaired t-test.

Figure 7

Correlation of immune responses with viremia. Innate, cell-mediated and humoral immune responses were correlated with peak viremia to determine if specific immune responses could be responsible for control of viremia. Shown are innate immune responses by CD56+CD3- and CD56+CD3+ NK cells in PBMC and spleen (A), cell-mediated responses by CD4+ and CD8+ T cells in draining lymph node and LPL (B), and humoral response using endpoint Env and Gag titers (C). Spearman correlations are shown, with p-values indicated.