Differences in time of virus appearance in the blood and virus-specific immune responses in intravenous and intrarectal primary SIVmac251 infection of rhesus macaques; a pilot study
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HIV-I can be transmitted by intravenous inoculation of contaminated blood or blood product or sexually through mucosal surfaces. Here we performed a pilot study in the SIVmac251 macaque model to address whether the route of viral entry influences the kinetics of the appearance and the size of virus-specific immune in different tissue compartments.
For this purpose, of 2 genetically defined Mamu-A*01-positive macaques, 1 was exposed intravenously and the other intrarectally to the same SIVmac251 viral stock and virus-specific CD8+ T-cells were measured within the first 12 days of infection in the blood and at day 12 in several tissues following euthanasia.
Virus-specific CD8+ T-cell responses to Gag, Env, and particularly Tat appeared earlier in the blood of the animal exposed by the mucosal route than in the animal exposed intravenously. The magnitude of these virus-specific responses was consistently higher in the systemic tissues and GALT of the macaque exposed by the intravenous route, suggesting a higher viral burden in the tissues as reflected by the faster appearance of virus in plasma. Differences in the ability of the virus-specific CD8+ T-cells to respond in vitro to specific peptide stimulation were also observed and the greatest proliferative ability was found in the GALT of the animal infected by the intrarectal route.
These data may suggest that the natural mucosal barrier may delay viral spreading. The consequences of this observation, if confirmed in studies with a larger number of animals, may have implications in vaccine development.
KeywordsLamina Propria Rhesus Macaque Intravenous Route Proliferative Ability Tetrameric Complex
List of abbreviations
(human immunodeficiency virus)
(high endothelial venules)
(peripheral blood mononuclear cells)
Infection with human immunodeficiency vims (HIV) elicits an acute retroviral syndrome characterized by fever, pharyngitis, lymphadenopathy, myalgia, rash, and headache [1,2,3]. Sexual transmission of HIV infection occurs mostly via the intestinal or vaginal mucosa but HIV-I is also effectively transmitted by the intravenous route  [5,6,7].
Recent studies have shown that the HIV-I or SIV virus rapidly penetrates vaginal, rectal, or oral mucosa attaching to and infecting primarily CD4+ T-cells where it replicates and consequently spreads to lymphoid tissue and systemic organs  [9,10,11,12].
Accumulating evidence has implicated virus-specific CTL in containing primary HIV/SIV infection and HIV-I/SIV-specific CD8+ CTL have been documented during the early weeks following infection, before a neutralizing Ab response is demonstrable  [14,15,16]. Despite the rapid dissemination of HIV-I by mucosal routes, productive mucosal transmission appears to be relatively inefficient and is estimated to occur once in 300 or more high-risk exposures . Cell-mediated immunity and direct killing by cytotoxic lymphocytes from the vagina and colon lamina propria may be an important factor in containing viral infection at the site of primary infection  [19,20].
Mucosal T lymphocytes appear to be functionally distinct from those present in the peripheral circulation. While activated T-cells reenter lymphoid tissues and preferentially accumulate at the site of the initial activation, memory T-cells migrate continuously and randomly, similar to naive T-cells  . The implication in terms of HIV infection is that, in the initial phase of an immune response, once primed, Ag-specific memory T-cells randomly enter and leave various lymphoid compartments but preferentially are retained in the lymphoid compartment where the antigen was presented at first  .
In humans, it is unfeasible to evaluate the immunological events that occur shortly after infection in the mucosal compartments. However, in the SIVmac251 macaque model, some of these issues can be addressed. SIVmac251 establishes persistent infection in rhesus macaques and causes an immunodeficiency syndrome closely resembling human AIDS  [26,27,28]. As in humans, the clinical course of SIVmac251 infection varies considerably among macaques. Recent evidence from our lab suggests that macaques that express the major histocompatibility class I Mamu-A*01 molecule restrict SIVmac251 replication following intrarectal exposure, as reported for HIV-I-infected individuals that express the HLA B*5701 , further validating this animal model of HIV-I infection. In this model, virus strain, dose, and especially route of infection can be defined and host-virus interactions under different conditions can be assessed. Here we used genetically defined Mamu-A*01 rhesus macaques to study the extent of virus-specific CD8+ T-cell response and the trafficking of lymphocytes to the gut during the first 12 days of intrarectal or intravenous transmission of the same stock of SIVmac251.
Animals and procedure
Two female Mamu-A*01-positive macaques were involved in this study: animal 817 that was infected by undiluted SIVmac251/561 stock virus (R. Pal et al., unpublished data) by the intrarectal route and animal 819 by the intravenous route with a 1:3000 dilution of the same viral stock. Blood was drawn before infection and at days 4, 8, and 12 after inoculation with the virus. Animals were sacrificed at day 12 postinfection. The spleens and hilar, axillary, mesenteric, iliac, and inguinal lymph nodes as well as ileums, jejunums, and colons were collected in RPMI medium containing 10% FCS+ penicillin/streptomycin.
Isolation of tissue lymphocytes
Mononuclear cells were isolated from peripheral blood (PBMC), lymph nodes, spleens, and intestines. Mononuclear cells from spleens and lymph nodes were isolated by mechanical dissociation of the tissue and consecutive Ficoll gradient centrifugation. Tissues from ileums, jejunums, and colons were treated with 1 mM DTT (ICN Biomedicals, Aurora, OH) for 30 minutes followed by incubation in calcium/magnesium-free HBSS with EDTA (Life Technologies, Baltimore, MD) 4 times 1 hour with stirring at room temperature to remove the epithelial layer. At this stage, pieces of tissue were fixed in 10% neutral formalin and embedded in paraffin and sections were cut and stained with H&E. Microscopic examination was performed to ensure that all of the epithelium was removed and the lamina propria was intact.
Further, tissues were cut into smaller pieces and incubated at 37°C in Isocove's medium supplemented with 10% fetal calf serum and penicillin/streptomycin containing 400 U/ml Collagenase D (Boerrhinghem & Mannheim GmbH, Mannheim, Germany) and 25 U/ml DNAse (Worthington Biochemical, Lakewood, NJ) for 2–3 hours. The mononuclear cells were isolated from the supernatant containing dissociated cells by Percoll gradient centrifugation.
Fresh mononuclear cells were directly stained with PE-conjugated tetrameric complexes for Gag 181, Env 622, and Tat 28. PerCP-conjugated anti-CD3e (Pharmingen, San Diego, CA) and FITC-conjugated anti-CD8 (Becton Dickinson, San Jose, CA) were used in conjunction with the tetrameric complexes. In addition, cells were stained with a combination of anti-CD3 PerCP, anti-CD8 FITC and anti CD4 PE (Becton Dickinson, San Jose, CA) . To assess their proliferative ability and to confirm the specificity of tetramer staining of ex vivo CD3+ CD8+ T-cells, lymphocytes were cultured at a concentration of 3 × 106/ml in RPMI enriched with 10% human serum with addition of 10 μg/ml of the appropriate peptide and 20 U/ml of IL-2 for 7 days. Staining with tetrameric complexes was done afterward as described above.
Briefly, 5 × l05 lymphocytes isolated by Ficoll diatrizoate or Percoll gradient centrifugation were incubated with tetrameric complexes as previously described  and/or selected Ab for 30 minutes at room temperature. After washing the cells twice in Dulbecco's phosphate buffered saline supplemented with 2% FCS and fixation in 1% paraformaldehyde (Ph = 7.4), samples were analyzed by flow cytometry using FACScalibur (Becton Dickinson, San Jose, CA) instrument.
Viral load (NASBA)
SIVmac251 viral RNA copies in plasma was quantified by nucleic-acid-sequence-based amplification . Briefly, RNA extracted from plasma was subjected to isothermal amplification with SIVmac251-specific primers. A portion of the SIV wild type Gag gene was used to generate internal control. NASBA amplification products were detected by using chemiluminescence-based probe hybridization system. The assay can measure accurately tenfold changes and is functional over a dynamic range spanning at least 104–107 copies.
Kinetics of viral appearance and CD4+ T-cell decrease in the blood
Since animal 817 received three thousandfold more virus than animal 819, these data suggest that the kinetics of viral appearance in the blood in primary SIVmac251 infection may depend on the route of challenge rather than dose.
Virus-specific CD8+ T-cell response in the blood
Virus-specific CD8+ T-cells were quantitated in the blood of both animals at days 0, 4, and 12 postinfection using 3 tetrameric Mamu-A*01 molecules complexed with the SIVmac Gag 181, Env 622, and Tat 28 peptides (T.M. Alien et al., submitted).
In the blood, virus-specific tetramer-binding CD3+ CD8+ T-cells appeared earlier in macaque 817, exposed to SIVmac251 by the intrarectal route, than in 819 for all the 3 antigens studied (Figs. 1B and 1D). Overall, the size of the virus-specific CD8+ T-cell response in the blood was higher in animal 817 and, interestingly, a large population of Tat-specific CD8+ T-cells appeared in the blood as early as day 4, whereas in the same animal CD8+ T-cells specific to Gag 181 and Env 622 peaked later (Fig. 1). These data are consistent with previous observations on the kinetics of appearance of virus-specific CD8+ T-cells that recognize the Tat 28 epitope and underscore the importance of the immune response to this early SIVmac251 protein [36,37].
Absence of CD4+ T-cell depletion in the GALT of Mamu-A*01 macaques
Quantitation of ex vivoCD8+ T-cell response in tissues
Illiac sublumbar lymph node
Axillar lymph node
Hilar lymph node
Mesenteric lymph node
Inguinal lymph node
Jejunum lamina propria
Ileum lamina propria
Colon lamina propria
Differential proliferative ability of tetramer-binding CD8+ cells in tissues of animals 817 and 819
The impact that the route of viral entry of HIV/SIV may have on the virus-specific immune response and control of viral replication to it is not yet fully understood. Here we conducted a small pilot study using genetically defined Mamu-A*01 macaques to study the vims-specific immune response following parenteral or rectal exposure to SIVmac251 This study was designed to begin to investigate the specificity of the immune response and its kinetics as well as the kinetics of viral replication according to the site of primary exposure and viral entry.
Within the limitation of the small number of animals used here, the mucosal barrier delayed the appearance of virus in the plasma whereas the decrease in the absolute CD4+ T-cell counts was equivalent. In addition, in contrast to other reports  , depletion of CD4+ T-cells in the GALT at day 12 postinfection was not observed in either animal regardless of the route of challenge. It is unclear what is the reason for this finding. It is possible that both animals could have controlled the infection or that the tissues were examined too early and that depletion would have occurred at a latter stage of the infection.
The finding of a higher percentage of tetramer-positive CD8+ T-cells in tissues of macaque 819, infected intravenously, than in animal 817, infected intrarectally, may reflect a higher rate of viral replication in tissues of this animal at the time of sacrifice. Of interest, the intrarectal route of infection resulted in faster kinetics of appearance of virus-specific CD8+ T-cells, particularly in the Tat epitope in the blood, indicating the importance of this immune response early in infection. In the same animal, a higher number of tetramer-specific CD8+ T-cells able to proliferate in response to all epitopes tested (Gag 181, Tat 28, and Env 622) was found in jejunum lamina propria than in other tissues. Unfortunately, at present, it is not possible to assess whether the differences in the proliferative ability of these cells reflect functional status and/or differentiation differences (effector versus memory) or others, in part because markers to define accurately these cells in rhesus macaques are not available.
In conclusion, this study, within the limitation of the small number of animals used, appears to suggest that exposure by the mucosal site, a natural barrier to pathogens, delays viral appearance in the blood. The differences in the relative percentage of homing markers may not necessarily reflect a true qualitative difference in the overall lymphocyte trafficking related to the mode of viral encounter and may depend on the time of analysis, since the kinetic of viral replication was delayed in the macaque exposed intrarectally. Nevertheless, these data demonstrate that a short window of opportunity to contain viral infection following mucosal exposure exists and that potentiating the effectiveness of the mucosal natural barrier by local immunization may further limit or halt viral replication. In fact, in a previous study, we have observed that it appears to be easier to protect vaccinated macaques by intrarectal exposure than intravenous exposure to SIV251 . In particular, since a CD8+ T-cell response to Tat appears to be the earliest, this protein may be a key component of a preventive vaccine, as suggested by other [36,37,43].
We thank John D. Altman and David Watkins for providing the SIV251 CD8+ T-cell epitope sequences and the corresponding tetramers, T. M. Allen for peptides, and Steven Snodgrass for editorial assistance.
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