Persistence of pathogenic challenge virus in macaques protected by simian immunodeficiency virus SIVmacDeltanef.

Live attenuated simian immunodeficiency virus (SIV) is the most efficient vaccine yet developed in monkey models of human immunodeficiency virus infection. In all successful vaccine trials, attenuation was achieved by inactivating at least the nef gene. We investigated some virological and immunological characteristics of five rhesus macaques immunized with a nef-inactivated SIVmac251 molecular clone (SIVmac251Deltanef) and challenged 15 months later with the pathogenic SIVmac251 isolate. Three animals were killed 2 weeks postchallenge (p.c.) to search for the challenge virus and to assess immunological changes in various organs. The other two animals have been monitored up for 7 years p.c., with clinical and nef gene changes being noted. The animals killed showed no increase in viral load and no sign of a secondary immune response, although the challenged virus was occasionally detected by PCR. In one of the monkeys being monitored, the vaccine virus persisted and an additional deletion occurred in nef. In the other monkey that was monitored, the challenge and the vaccine (Deltanef) viruses were both detected by PCR until a virus with a hybrid nef allele was isolated 48 months p.c. This nef hybrid encodes a 245-amino-acid protein. Thus, our results show (i) that monkeys were not totally protected against homologous virus challenge but controlled the challenge very efficiently in the absence of a secondary immune response, and (ii) that the challenge and vaccine viruses may persist in a replication-competent form for long periods after the challenge, possibly resulting in recombination between the two viruses.

Viral load and neuropathology in the SIV model.

To investigate neuropathological processes involved in HIV infection, a longitudinal analysis of central nervous system (CNS) changes was performed using the SIV-infected macaque model. Five animals were studied during the early phase and 13 during the asymptomatic and symptomatic phases. Histopathological analyses were performed on one cerebral fixed hemisphere whereas on the other frozen hemisphere in situ hybridisation, immunohistochemistry and RT-PCR were performed. Viral load was quantified by in situ hybridisation, CD4 and CD8 T cell infiltration by immunohistochemistry and mRNA cytokine expression (IL1beta, IL2, IL6, TNFalpha, IFNgamma and TGF-beta1) by semiquantitative RT-PCR. As reported for HIV-infected humans, the neuropathological analysis of SIV infected animals revealed four distinct lesion profiles: minimal changes, early encephalitis, leukoencephalopathy and encephalitis. No relationship was found between neuropathological findings, numbers of SIV replicating cells and T cell infiltration. CNS infection was found to be an early event characterised by glial activation, an increase in the level of IL1beta, TNFalpha and IL6 mRNA expression. During the asymptomatic and symptomatic phases, IL6 and IL1beta mRNAs increase coincided with gliosis and the development of myelin lesions. The absence of relationship between neuropathological findings and viral load suggests that cerebral lesions are caused by an indirect mechanism. Inflammatory cytokine pattern associated with severe lesions show the key role of glial activation in the SIV neuropathological process.

[Contribution of animal models in the understanding of AIDS encephalopathy]. / Apport des modèles animaux dans la compréhension de l'encéphalopathie SIDA.

The neuropathology associated with HIV (Human Immunodeficiency Virus) infection is one of the major complications of this disease. The virological and cellular mechanisms by which HIV infection induces motor and cognitive disorders remain unknown. This lack of understanding of the pathophysiology is partly due to the difficulty of experimental analysis in man because only post-mortem samples from terminal phases of the disease and cerebrospinal fluid samples are available. Two animal models, very closely resembling human HIV infection, are available: the cat model infected by FIV (Feline Immunodeficiency Virus) and the macaque model infected by the SIVmac (Simian Immunodeficiency Virus) which have enabled us to conduct a longitudinal study of encephalopathy during primo-infection and the asymptomatic and pre-AIDS (Acquired Immune Deficiency Syndrome) phases. In the cat-FIV model, which presents the advantage of being non-infectious to man, and therefore easier to manipulate, it was shown that infected cats develop behavioural abnormalities and a neuropathology which resemble HIV dementia. Central nervous system lesions induced by FIV are similar to those of HIV infection apart from the absence of multinucleated giant cells. This model was used to analyse the relationship between CNS lesions and the viral load of the brain and showed that the severity of the lesions contrasted with a low viral load. The pathophysiology of SIVmac infection in the rhesus macaque is almost identical to human infection with a more rapid course, since the duration of the asymptomatic phase is 6 months to 5 years, depending on the animal. We studied the relationship between lesions, viral load and cytokine production (IL-1 beta, IL-2, IL-6, TNF alpha, INF gamma, TGF-beta 1) within the CNS. Our results show early, low-grade and constant infection of the brain. The dissociation between the viral load and the lesions observed is our favour of an indirect mechanism for the pathogenesis of these lesions. The relationship between lesions and the cytokine profile studied shows the importance of glial cells in the pathogenesis of the lesions.

Interferon-gamma expression in macaque lymph nodes during primary infection with simian immunodeficiency virus.

Simian immunodeficiency virus (SIV) replication is rapidly downregulated in the lymph nodes (LN) of rhesus macaques after the acute stage of primary infection. The aim of this study was to evaluate a possible role of interferon-gamma (IFN-gamma) in the control of SIV replication. IFN-gamma expression was analysed by in situ hybridization in the LN of rhesus macaques that were inoculated either with a high dose or with a low dose of the pathogenic isolate SIVmac 251. The kinetics of IFN-gamma induction in LN was found to follow that of SIV replication. However, the number of IFN-gamma expressing cells was not proportional to the number of infected cells. IFN-gamma expression in LN was further quantified by competitive RT-PCR. The number of IFN-gamma mRNA molecules in LN was high for the animals of the high dose group. In the low dose group, the IFN-gamma copy number varied over 2 log10 units and was particularly low for the animals that had a high and persisting antigenaemia. The analysis of a total of 10 animals inoculated with a low dose of virus showed an inverse correlation between IFN-gamma expression in LN and peak antigenemia (P < 0.01). This study provides evidence for a marked individual variability in the IFN-gamma response to primary SIV infection and supports the notion that IFN-gamma production is inhibited at an early stage in animals that harbour a high viral load.

The relationship between the interferon alpha response and viral burden in primary SIV infection.

The interferon alpha (IFN-alpha) response of rhesus macaques was investigated during primary infection with pathogenic and attenuated simian immunodeficiency virus (SIV). IFN-alpha was detected in the serum of animals as early as day 4 after inoculation of SIVmac251, but remained barely detected in animals infected with the attenuated virus SIVmac251 delta nef. The peak of IFN-alpha secretion preceded that of antigenemia in animals infected with pathogenic virus, indicating that the IFN-alpha response did not prevent viral spread. In addition, elevated levels of IFN-alpha in the serum after the acute stage of infection was associated with persisting antigenemia. The analysis of lymph nodes (LNs) by in situ hybridization showed that, similar to the results obtained with peripheral blood, the induction of IFN-alpha in lymphoid organs was rapidly detected in animals infected with the pathogenic virus, but remained very limited in animals infected with the attenuated virus. Quantitation of the hybridization signal indicated that IFN-alpha-producing cells were numerous in the LNs of animals that had a high viral burden. Taken together, these findings indicate that the IFN-alpha response is unable to contain the initial burst of SIV replication.

Cytokine patterns and viral load in lymph nodes during the early stages of SIV infection.

To elucidate the initial pathogenic events in lymphoid organs, the major reservoir of virus in HIV infection, follow-up of viral load and cytokine mRNA production was performed in the rhesus macaque SIV mac251 model. In the first experiment, lymph nodes (LN) obtained from animals sacrificed at early time points following intravenous (i.v.) inoculation with a high viral dose, showed that the production of cytokine mRNA in LN (IFN gamma, IL1 beta, IL2, IL4, IL12p40, IL6, IL10, TNF alpha and TNF beta) was correlated with viral replication and persisted during the two months post-inoculation (p.i.). In the second experiment, LN were sequentially analysed in two groups of animals receiving i.v. a high or a low viral dose. The initial higher local production of IFN gamma mRNA in LN was associated with weak viraemia, the capacity of the host to decrease productive infection in LN measured at one and two months p.i., and slow disease progression.

Limited viral spread and rapid immune response in lymph nodes of macaques inoculated with attenuated simian immunodeficiency virus.

A comparative study was undertaken to characterize the very early events that distinguish attenuated and pathogenic simian immunodeficiency virus (SIV) infections. Three rhesus macaques were inoculated with the attenuated SIVmac 251 delta nef virus, and three others with a virus of intermediate phenotype, SIVmac 239 nef stop. They were compared to four macaques inoculated with the pathogenic SIVmac 251 isolate. Lymph nodes (LN) taken between 7 days and 2 months postinoculation were analyzed for SIV expression by in situ hybridization. During acute infection, SIV 21 delta nef infected 1 to 1.5 log10 fewer cells in LN tissue than the pathogenic SIV 251 isolate. The reduction was more marked in the blood, as SIV 251 delta nef infected 2 to 3 log10 fewer PBMC than the isolate and did not yield detectable antigenemia. Morphometric measurements showed that the development of germinal centers (GC) was more rapid in the delta nef infection, which led to a more efficient trapping of viral particles, and could account for antigenemia clearance. The SIV 239 nef stop clone reverted to a nef+ genotype at Week 2, but induced a lower viral burden than a directly pathogenic virus. The kinetics of GC development was rapid, indicating that SIV 239 nef stop induced an immune response similar to that seen in attenuated infection. This study provides evidence that attenuated SIV elicits a more rapid immune response than pathogenic SIV and suggests that an early immunosuppressive episode may facilitate the dissemination of pathogenic SIV.