Readily acquired secondary infections of human and simian immunodeficiency viruses following single intravenous exposure in non-human primates

Peter ten Haaft, Ernst J. Verschoor, Babs Verstrepen, Henk Niphuis, Rob Dubbes, Wim Koornstra, Willy Bogers, Brigitte Rosenwirth and Jonathan L. Heeney

Department of Virology, Biomedical Primate Research Centre, PO Box 3306, 2280 GH Rijswijk, The Netherlands

Correspondence
Jonathan L. Heeney
heeney{at}bprc.nl


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Accumulating evidence suggests that exposed individuals may acquire multiple human immunodeficiency virus (HIV) infections more frequently than originally believed. As a result, circulating recombinant forms of HIV are emerging that are of particular concern in the AIDS epidemic and HIV vaccine development efforts. The aim of this study was to determine under what conditions secondary or superinfections of HIV or simian immunodeficiency virus (SIV) may be acquired under controlled settings in well-defined, non-human primate models. Retrospective analysis of macaques that had acquired apparent immunity upon infection with a defined attenuated SIVmac strain revealed that eight out of eight animals that were secondarily exposed to a new virus variant became infected with the new virus strain, but at low levels. Interestingly, similarly high frequencies of secondary infections were observed after early (4 months), as well as late (5 years), exposure following primary infection. As possible causes of susceptibility to secondary infections, perturbations in the immune system associated with exacerbated infections were then investigated prospectively. Results revealed that short-term immune-suppression therapy did not increase susceptibility to secondary infections. Taken together, data suggested that neither early- nor late-exposure immune-suppressive events following primary infection accounted for the observed high incidence of secondary infections. With HIV-1, the question of whether secondary infections with very closely related viral variants could occur in the chimpanzee model was addressed. In both animal models, secondary infections were confirmed, notably with relatively closely related SIVmac or HIV-1 strains, following a single exposure to the secondary virus strain. These findings reveal that secondary lentiviral infections may be acquired readily during different stages of primary infection, in contrast to co-infections, which are acquired at the moment of initial infection.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
New human immunodeficiency virus (HIV) variants that reveal mosaic HIV-1 genomes as a result of recombination have emerged in the recent AIDS epidemic (Cornelissen et al., 2000; Laukkanen et al., 2000; Piyasirisilp et al., 2000; Ramos et al., 2003; Takebe et al., 2003; Tscherning-Casper et al., 2000; Yang et al., 2003). These variants, termed circulating recombinant forms (CRFs), have been identified in patients from several continents. In specific areas, such as western Africa, CRFs are commonly found (Peeters et al., 2003) and those with greater viral fitness in these human populations are overtaking previously circulating HIV strains in these regions. The high mutation rate, leading to the evolution of viral variants (antigenic drift), was seen as a major obstacle to the development of an HIV vaccine (Sasaki & Haraguchi, 2000). However, the emergence of CRFs represents an additional HIV vaccine challenge, with major antigenic shifts in the circulating HIV population (Mooij & Heeney, 2001). In this context, scenarios that may facilitate the emergence of CRFs are important to understand.

The generation of CRFs requires the co-infection of a single cell by more than one variant of HIV (Magiorkinis et al., 2003). Until recently, it was believed that infection of an individual with more than one variant of HIV was a rare event. However, accumulating single-case reports suggest that secondary infections may occur more commonly than previously believed (Altfeld et al., 2002; Jost et al., 2002; Koelsch et al., 2003; Ramos et al., 2002). The understanding that the immune response to primary infection can afford protection from subsequent infection by other HIV variants was largely based on observations from live attenuated SIVmac vaccine studies that were performed in non-human primates (Desrosiers, 1998). It is currently not known when an infected individual may become infected with another, distinct variant of HIV. Many suggest that multiple variants are transmitted at the time of initial infection, but only become dominant later, thus resembling a secondary infection. Alternatively, others suggest that secondary infections are acquired a long time after initial infection, when an individual's immune system begins to deteriorate. These questions are difficult to address in humans when the date, route, dose and viral composition of the inoculum are unknown. Given these difficulties in studying the events that accommodate secondary HIV infections in humans, we utilized two non-human primate HIV/AIDS models, the rhesus macaque and chimpanzee, to elucidate the circumstances under which secondary or multiple lentiviral infections may be acquired. Here, we set out to determine whether secondary infections could be acquired in the presence of a persistent, asymptomatic HIV infection prior to the onset of virus-induced immune suppression. For this reason, we turned to the use of live attenuated SIV infection in macaques and HIV-1 in chimpanzees to model variations in this scenario in humans as closely as possible. In this setting, we were able to control important variables such as the timing of exposure, the route and the viral composition and characteristics of the primary and secondary inocula.

We first examined whether secondary infections were more likely to occur relatively early (4 months) or late (>5 years) following primary infection. Adaptive anti-HIV immune responses are induced soon after infection, but require time to mature. In addition, it has been suggested that subclinical immune suppression can occur during the early asymptomatic period (Miedema et al., 1988; Teeuwsen et al., 1990). Similarly, several years following HIV infection, immune responses begin to decay as individuals begin to progress to AIDS. In a second study, we set out to determine whether insufficient or compromised immunity could lead to increased susceptibility to secondary infections. To address the question of whether healthy infected individuals who controlled virus replication could acquire secondary infections, even with very closely related variants, we turned to the HIV-1 model in chimpanzees. Both model systems confirmed that, in contrast to simultaneous co-infection occurring during a single exposure, true secondary infections could be acquired readily at early, as well as later, time points during the asymptomatic period.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Experimental design and study groups.
Three experimental settings were studied, using either captive-bred rhesus macaques (Macaca mulatta) or chimpanzees (Pan troglodytes). All experiments were performed following ethical approval and according to strict international guidelines for housing and use of primates in biomedical research.

Study 1 comprised a retrospective analysis of three groups of four age-matched, outbred rhesus macaques (Table 1). Groups 1A (animals 1RZ, 1SV, 1XY and 1YC) and 1B (animals 1YO, 1YR, 2BR and 1VY) were infected intravenously (i.v.) with 16x103 TCID50 SIVmac251-BK28, containing an attenuating deletion in nef (Kornfeld et al., 1987). Seven out of eight infected animals remained asymptomatic for >5 years. Four other animals (group 1C) were used as naïve controls for secondary exposure. All three groups were subjected to secondary i.v. exposure with SIV8980, as described previously (Heeney et al., 1994). SIV8980 was derived from SIVB670 by four subsequent in vivo passages of late-stage disease virus. The challenge stock, SIV8980, was cultivated on autologous peripheral blood mononuclear cells (PBMCs) from the fourth passaged macaque (8980), which developed end-stage AIDS within 1 month of infection. SIV8980 characteristically causes rapid loss of T-helper and T-memory cells, combined with high steady-state levels of plasma viraemia. The pathology of acute AIDS induced by SIV8980 closely resembles that observed after a chronic disease course (Holterman et al., 1999). DNA sequence identity between SIVBK28 and SIV8980 is 87 % in gag, 82 % in pol and 83 % in env (Holterman et al., 2001). Group 1A was subjected to secondary exposure with SIV8980 4 months following primary infection with SIVBK28; group 1B was subjected to secondary exposure 5 years following primary infection.


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Table 1. Investigated causes of increased susceptibility to secondary infections

Study 1, early (4 months) versus late (5 years) secondary exposure post-primary infection (rhesus macaques); study 2, secondary exposure following immune suppression during primary infection (rhesus macaques); study 3, secondary exposure of long-term asymptomatic chimpanzees with a highly related HIV-1. n, No. animals in each group.

 
Study 2 was designed prospectively to determine the effect of potent immune suppression on susceptibility to secondary infection. The study consisted of two groups of five age-matched, mature rhesus macaques, whilst two animals were used as naïve controls for the secondary exposure (Table 1). Groups 2A (animals R905, P962, R187, R497 and R420) and 2B (animals R535, R431, R708, R903 and P970) were inoculated i.v. with a molecular derivative of SIVmac239 (SIV{Delta}nef) with a specifically engineered deletion in nef (Daniel et al., 1992). Group 2B was immune-suppressed (starting at week 12 post-primary infection), effectuated by five subcutaneous injections of 60 mg anti-thymocyte globulin (ATG) kg–1 given at 2-day intervals (days 0, 2, 4, 6 and 8). Thirty-eight weeks after SIV{Delta}nef infection of groups 2A and 2B, all 12 animals were challenged with 50 MID50 SIV8980, the same secondary virus strain as in the first study. Rhesus monkeys were euthanized if they developed clinical, haematological and virological evidence of AIDS and a complete necropsy was performed to confirm the diagnosis of AIDS.

Study 3 consisted of two groups of mature chimpanzees (Table 1). Group 3A (Ch-Bu and Ch-Ma) was infected i.v. with HIV-1Lai. After a 12-year period of HIV-1Lai infection, the animals were subjected to secondary exposure with 100 TCID50 of a cell-free stock of the primary clade B isolate HIV-1Han2 (Sauermann et al., 1990). Ch-On and Ch-Su served as retrospective naïve controls (group 3B) and were infected i.v. with 10 and 100 TCID50, respectively (Bogers et al., 1998). DNA sequence identity between HIV-1IIIB and HIV-1Han-2 is 95 % in gag, 97 % in pol and 89 % in env.

Tissue samples and fluorescence-activated cell sorter (FACS) analysis.
PBMCs were isolated from heparinized blood as described previously (Mooij et al., 2000). Cells were either used immediately or cryopreserved for later analysis. Cell suspensions were prepared from peripheral lymph-node biopsies and, at necropsy, from spleen, bone marrow and thymus. Mononuclear cells from the cell suspensions were prepared as for PBMCs. To monitor CD3+, CD4+, CD8+ and CD4+/CD29+ subsets of PBMCs, FACS analysis was performed essentially as described previously (Mooij et al., 2000).

PCR assays.
Genomic DNA was isolated from mononuclear cells by using proteinase K/Triton X-100-based lysis followed by ethanol precipitation. For diagnostic purposes, two different PCR assays were carried out on each DNA sample, using nested gag and LTR primer sets for SIVmac (Bogers et al., 1995) and nested gag and env-V3 primer sets for HIV-1 (ten Haaft et al., 1995). The sensitivity of these PCR assays was established at one copy of proviral DNA in 1 µg genomic DNA (the equivalent of 1·5x105 cells). Viral RNA was purified as described previously (ten Haaft et al., 1998). Reverse transcription and amplification of the outer-primer reaction were carried out in a single-step protocol by using the Superscript One-Step RT-PCR system (Invitrogen). From the RT-PCR, 5 µl product was transferred to an inner-primer reaction mixture. For inner PCR, the same reaction conditions and cycle protocol were used as for the inner PCR on DNA. To discriminate between different virus strains, sets of restriction enzymes were selected to give distinct RFLP patterns. Digestion products were separated by gel electrophoresis in 3·5 % Metaphor agarose (FMC Bioproducts), stained with ethidium bromide and analysed.

Virus load assays.
A quantitative competitive (QC) RT-PCR was used to determine the SIV virus load in plasma (ten Haaft et al., 1998). The sensitivity of the QC RT-PCR assay was established at 40 copies RNA (ml plasma or serum)–1. The HIV-1 virus load in plasma of chimpanzees was measured by using the Amplicor HIV-1 Monitor system, version 1.5 (Roche Diagnostics).

To calculate the virus load of the PBMCs, a QC DNA PCR was used. To each PCR, 1 µg genomic DNA was added, together with a known amount of internal-standard DNA to monitor the amplification efficiency. The detection procedure and calculation of the virus load were identical to the QC RT-PCR protocol (ten Haaft et al., 1998).

Heteroduplex mobility assay (HMA).
To determine the presence of one or more viral variants, heteroduplex formation of PCR products and their analysis were performed as described by Delwart et al. (1995) with minor modifications. For HMA, viral RNA was isolated from plasma as described above. For HIV-1-infected chimpanzees, outer primer set ED3/ED14, amplifying a 2 kb fragment from the first exon of rev to the gp41-coding region of env (nt 5956–7960 of the HIV1HXB2 genome, GenBank accession no. K03455) and inner primer set ED5/ED12, amplifying a 1·2 kb fragment spanning the V1–V5 coding region of gp120 (nt 6556–7822 of the HIV-1HXB2 genome) were used (Delwart et al., 1995). For SIV-infected rhesus macaques, two nested primer sets were used within the 5' LTR/gag and env/nef regions. The outer SIV LTR/gag set amplified a 776 bp fragment from the 5' LTR to gag (nt 699–1475 of the SIVsmmH4 genome, GenBank accession no. X14307); the inner SIV gag/LTR set amplified a 621 bp fragment from the 5' LTR to the p24 nucleocapsid (nt 825–1446 of the SIVsmmH4 genome). The outer SIV env/nef set amplified a 1·8 kb fragment from the end of gp120 to nef (nt 8007–9834 of the SIVsmmH4 genome); the inner SIV env/nef set amplified a 1·6 kb fragment from the start of gp41 to nef (nt 8144–9786 of the SIVsmmH4 genome). Probes were generated from control animals at the peak of primary viraemia. To identify heteroduplexes formed by quasispecies of the infecting strain and to monitor PCR co-amplification of both primary and secondary virus, PCR products from the samples were subjected to self-heteroduplex formation, in addition to sample–probe heteroduplexes.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Effect of the time point of secondary SIV exposure in macaques
To determine whether secondary infections possibly occurred early or late after primary infection, we retrospectively analysed samples from a previous study (Heeney et al., 1994), from which we had preliminary evidence of secondary SIV infection (ten Haaft et al., 1996). This study consisted of 12 animals, four naïve controls and eight animals infected with SIVmacBK28. All eight animals had controlled the primary infection and were then exposed to a secondary variant, SIV8980, 4 months (four animals) or 5 years (four animals) following primary infection. In archived blood samples, we searched for evidence of dual infection by discriminative PCR. Provirus of the SIV8980 strain was detected at one or more time points in all animals that received the early secondary exposure (group 1A). In three of the four animals (1SV, 1XY and 1YC), the presence of SIV8980 provirus was confirmed at multiple time points, concurrent with the primary SIVmac variant. In animal 1YC, SIV8980 even became the most predominant virus (Table 2), best described by the term superinfection. In the late (5 year) secondary-exposure group (1B), SIV8980 provirus was found in three out of four animals at multiple time points. Furthermore, in all animals from groups A and B, SIV8980 RNA was detected in serum (Table 2, groups 1A and 1B), which is clearly indicative of secondary co-infection. These results demonstrate that, in both scenarios, the secondary virus persisted at least as provirus, whilst intermittently detectable viral RNA in plasma demonstrated active replication of the secondary virus. A third technique, HMA, was used to corroborate our findings in the three animals that eventually progressed to disease (1SV, 1YO and 1VY; Table 3). In animals 1SV and 1VY, persistence of SIV8980 was confirmed, whereas in animal 1YO, only the primary virus was detected.


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Table 2. Analysis for primary- and secondary-exposure virus by discriminative PCR

Analysis results from three studies are combined. – (blue shading), Neither primary (P)- nor secondary (S)-exposure virus was detected; +, virus was detected, but no discrimination between primary- and secondary-exposure virus could be made. No shading, no sample possible.

 

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Table 3. HMA analysis to corroborate discriminative PCR results

PCR products for HMA were generated by nested RT-PCR on viral RNA from plasma or serum. He, Heteroduplex; Ho, homoduplex; Qs, quasispecies.

 
Effect of immune suppression on establishment of secondary infections
In the second study, ten animals were infected with another, related SIV strain harbouring an engineered nef deletion. Following the establishment of primary infection, animals were studied for susceptibility to secondary infection in the presence or absence of compromised immunity induced by ATG treatment. Group 2A (Table 1) was exposed to SIV8980 at week 38 post-primary infection. Infection with SIV{Delta}nef resulted in plasma viraemia, with a peak titre of between 1·5x104 and 3·9x105 RNA equivalents ml–1 at 2 weeks post-infection (Fig. 1a). In group 2A, plasma RNA levels declined in three animals, P962, R497 and R420, to levels below 3x102 RNA equivalents ml–1, or even became undetectable. The other animals of group 2A, R905 and R187, had persistently elevated RNA levels (Fig. 1a). In addition, these two animals had the lowest percentage of CD4+/CD29+ T cells in the PBMC population (data not shown).



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Fig. 1. Comparison of plasma viral RNA loads (RNA equivalents ml–1) of rhesus macaques following primary infection with SIV{Delta}nef and subsequent secondary exposure with SIV8980 (study 2). (a) Primary infection with SIV{Delta}nef at week –38; (b) primary infection with SIV{Delta}nef and immune-suppressed for the duration of week –26; (c) naïve controls. All animals were inoculated with SIV8980 at week 0.

 
Animals of group 2B were immune-suppressed by five s.c. injections of ATG at days 0, 2, 4, 6 and 8, starting at week 12 post-primary infection. The potent effect of ATG treatment was evident by a profound depletion of CD4+ and CD8+ T cells (data not shown). Following cessation of ATG treatment, a prompt return of the T-cell populations was observed. However, CD4+ and CD8+ counts did not return completely to normal levels; this phenomenon was also observed in humans treated with ATG to reduce the risk of graft-versus-host disease (Muller et al., 1997). Interestingly, during immune suppression, plasma RNA levels increased in all animals and then dropped to pretreatment levels following termination of ATG treatment (Fig. 1b).

As controls to monitor exposure to SIV8980, two naïve control animals (R231 and R378) (group 2C) were studied. Both animals developed peak plasma virus loads of >107 RNA equivalents ml–1 (Fig. 1c). Following seroconversion, steady-state levels reached as high as 108 RNA equivalents ml–1, whereas the level of circulating CD4+/CD29+ T cells in these animals declined progressively (data not shown). SIV8980 provirus was found in PBMCs, as well as lymph nodes, at all time points measured (Table 2). Due to clinical evidence of AIDS development, these two animals were euthanized at weeks 14 and 16 post-infection, respectively.

In group 2A (no immune-suppressive treatment), four of the five animals (P962, R187, R497 and R420) were found to have evidence of secondary SIV8980 infection, as determined by PCR on PBMCs, lymph-node DNA and plasma RNA samples (Table 2). At necropsy, these findings were confirmed by discriminative PCR on DNA derived from spleen, thymus and bone-marrow mononuclear cells (Table 2). Interestingly, animal R905, which had developed the highest SIV{Delta}nef plasma load (Fig. 1a) and had the highest proviral DNA levels prior to secondary exposure, was the only animal that resisted secondary exposure successfully (consistently negative for SIV8980 at all time points in all assays; Table 2) whilst remaining consistently positive for SIV{Delta}nef. A post-secondary exposure peak in plasma RNA level was seen in all animals (Fig. 1a), following which viral RNA levels in animals P962, R497 and R420 declined. Animal R187 maintained a high plasma virus load of between 106 and 107 RNA equivalents ml–1. The level of CD4+ T cells in the circulation of this animal declined progressively until euthanasia and correlated inversely with the high plasma RNA load. In the immune-suppressed group (2B), three of five animals (R535, R708 and P970) became infected with SIV8980 (Table 2), whereas two animals (R431 and R903) remained negative for SIV8980 and were only positive for SIV{Delta}nef. HMA analysis confirmed the discriminative PCR results (Table 3).

In total, seven of the ten secondary-exposed animals became infected with SIV8980. However, whilst no significant difference in susceptibility to secondary infections was found between these two groups, the transiently immune-suppressed group revealed three cases of overt ‘superinfection’. In these cases, the secondary variant became the dominant virus population in plasma, according to our working definition of superinfection. In contrast, only one case of superinfection occurred in the untreated group.

Secondary exposure of chimpanzees with a highly related HIV-1 isolate
To address the question of whether secondary infections could occur in healthy HIV-1-infected individuals, we turned to the HIV-1 chimpanzee model. In this species, the majority of animals remain asymptomatic and are able to control most HIV-1 infections, with either primary isolates or lab-adapted strains. The loosely used term ‘superinfection’ with distinct HIV-1 variants has been reported in chimpanzees following exposure with high doses of relatively unrelated viruses (Fultz, 2004; Levy, 2003). Based on these observations, we hypothesized that infection with closely related viral variants would be difficult to establish, due to host immune responses to shared epitopes generated by the primary infection.

In total, four chimpanzees were included in these studies. Two animals with asymptomatic HIV-1Lai infection were selected, having normal CD4+ T-cell numbers for >12 years (Table 1). Two others served as retrospective naïve infection controls (Bogers et al., 1998). All animals were exposed to the primary isolate HIV-1Han2 and all four animals became infected. Following secondary exposure, peak plasma viral RNA concentrations in both chronic, asymptomatic, HIV-1Lai-infected chimpanzees were 1–2 logs lower and, in one case (Ch-Ma), were delayed as compared with the naïve controls (Fig. 2). By 20 weeks, plasma virus RNA concentrations declined to <100 copies RNA ml–1 in three of the four animals. Most importantly, however, discriminative PCR on PBMC DNA from the two chronically infected, secondary-exposed animals revealed persistence of the secondary HIV-1Han2 isolate (Table 2, group 3A), even after plasma viral RNA loads had declined below levels of detection (Fig. 2). HMA results supported the discriminative PCR results (Table 3) that persistent co-infection with two distinct clade B isolates had occurred.



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Fig. 2. Comparison of plasma viral RNA loads (RNA equivalents ml–1) of chimpanzees following primary infection with HIV-1Lai and subsequent secondary exposure with HIV-1Han-2 (Ch-Bu and Ch-Ma) and naïve secondary-infection controls (Ch-On and Ch-Su). All animals were inoculated with HIV-1Han-2 at week 0.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Recently, several case reports of HIV-1 ‘superinfection’ in infected individuals have been documented (Altfeld et al., 2002; Jost et al., 2002; Koelsch et al., 2003; Ramos et al., 2002). Similarly, so-called superinfection has also been reported previously in chimpanzees receiving high doses of relatively unrelated viruses (Fultz, 2004; Fultz et al., 1987; Levy, 2003). Here, we reveal an important distinction between overt superinfection, in which a newly acquired variant becomes the predominant virus population in plasma, and the more insidious secondary infections, which are acquired after primary infection has occurred. Both scenarios need to be distinguished from multiple HIV-1 variants that may be acquired simultaneously upon initial exposure. Most significantly, our results reveal a new class of secondarily acquired lentiviral infections that are insidious and difficult to diagnose. These appear to be frequently acquired at various stages following primary infection and may persist, with the possibility of contributing to new recombinant variants (i.e. CRFs).

The present study set out to determine the various conditions under which secondary HIV-1 and SIV infections may be acquired by an infected host. Unexpectedly, by using multiple analyses and detailed follow-up, we demonstrated that secondary infections were acquired readily following intravenous exposure under various conditions. These data were confirmed in two different animal models with either pre-existing SIV or HIV-1 infection. In the first study in the SIV macaque model, we showed that, although primary infection was controlled, early or late exposure to secondary virus resulted in secondary infection of all animals. In one case, we felt confident about using the term ‘superinfection’ because of the overwhelming predominance of the secondarily acquired virus in plasma and tissues. Incidental failure of attenuated virus to protect against a secondary exposure has been described by others (Gundlach et al., 2000; Khatissian et al., 2001), but never to this extent. Three out of eight animals progressed to disease within the study period and the predominance of SIV8980 in two of these animals suggests strongly that they developed AIDS due to this secondary strain or a recombinant, as described previously (Gundlach et al., 2000; Wooley et al., 1997). In the second study, our observations were confirmed and extended by demonstrating that immune-suppressive treatment did not increase susceptibility to secondary infection in the model studied. Interestingly, animals with higher viral RNA loads were apparently less susceptible to secondary infections, suggesting that the establishment of secondary infection may rather be influenced by the number of available, uninfected target cells. Alternatively, in such circumstances, secondary infections may be prevented by local, innate-like mechanisms. In the immune-suppressed group, three animals demonstrated overt superinfection, in contrast to one animal in the untreated group. Apparently, short-term immune-suppressive treatment did affect the ability to control the secondary virus, but it did not increase susceptibility to secondary infection. Finally, studies with HIV-1 revealed that, despite the very close relatedness of the new variant, secondary infection occurred readily in chimpanzees. This result was surprising, as the primary infection with HIV-1Lai had been controlled successfully for >12 years.

These results in two different species demonstrate that secondary infections can be acquired readily within the animal models studied. The only correlation with reduced susceptibility to secondary infection was a high viral RNA load, probably high enough to facilitate disease progression (ten Haaft et al., 1998; Watson et al., 1997). HMA analysis did not reveal, but did not rule out, recombinants in plasma of these animals. If recombinants were present, they were probably less fit than those causing the initial infection. Previous studies in non-human primates, however, have reported recombinants with increased pathogenic potential (Mwaengo & Novembre, 1998; Gundlach et al., 2000; Wooley et al., 1997).

Parallels can be drawn with the live attenuated studies that have been carried out extensively in various macaque species over the last decade. The majority of live attenuated vaccine studies have reported ‘protection’, as the analyses often lacked data obtained from DNA PCR of PBMCs or, more importantly, lymph nodes or other potential reservoirs. In the original report, no proviral sequences of the challenge virus were detected in the four vaccinees, two of which were challenged with a homologous and two with a very closely related variant (Daniel et al., 1992). In most cases of heterologous challenge, the conclusions drawn have been in the context of protection from disease (observed as longer survival or extrapolated on the basis of lower plasma RNA loads and/or sustained CD4+ T-cell counts). Only a limited number of studies have reported incidental persistence of challenge virus (Wyand et al., 1999), although the risk of recombination between vaccine and challenge strain has been documented (Gundlach et al., 2000; Khatissian et al., 2001). In the majority of recent studies, follow-up after challenge included quantitative virus isolation and quantitative measurement of viraemia. When performing discriminative DNA PCR on PBMCs or lymph-node cells as post-challenge follow-up (Cranage et al., 1997; Nilsson et al., 1998; Sharpe et al., 1997; Titti et al., 1997), low-level secondary infections may have remained undetected. This may be due to the peak viraemia of the primary virus that generated high numbers of provirus-positive cells, whether capable of producing virus or not, causing a surplus of template bias in discriminative PCR assays. The possible correlates of protection of live attenuated vaccines have been investigated. High levels of {beta}-chemokines and antiviral CD8+ cell-suppressor activity have been hypothesized to be protective (Ahmed et al., 1999). Others reported strong cellular immune responses elicited by attenuated viruses (Johnson et al., 1997). In a study where no evidence of superinfection was detected, it was found that rechallenge did not alter cytokine profiles or frequency of activated PBMCs or generate anamnestic antibody responses. Perhaps the most important correlate of protection afforded by live attenuated vaccines is the replication capacity of the vaccine strain. Weak, poorly replicating (but safer) attenuated strains are less effective at protecting from superinfection than better-replicating strains (Desrosiers, 1998). It has been hypothesized that this mechanism of protection may be due to viral interferance or to triggering of as-yet-undefined innate responses (Stebbings et al., 2002). Our findings suggest that a revisit of previous studies (where HIV-1 or SIV was not detected in the context of superinfection) may reveal that infection actually did occur, as evidenced by challenge provirus in tissues. In such studies, discrimination may be made between low-level secondary infections (detected at the proviral level) and predominant superinfections that manifest as new dominant viral RNA at high levels in plasma.

Extrapolation of our findings to the human situation cannot be made without consideration of the restrictions of the models used. We exposed animals i.v. in a controlled setting with doses of uniform, defined virus strains. The dose of virus that i.v. drug users may be exposed to is difficult to define, but is likely to be lower than those used in preclinical studies. It is not unlikely that, during the course of asymptomatic HIV-1 infection, individuals will be exposed to other HIV-1 variants. However, to be able to conduct a study with an ethically appropriate number of animals and not to increase the study period extensively, we used a secondary virus that was more virulent than the primary virus. In addition, for the sake of reproducibility, inoculations were performed i.v. for both exposures. Obviously, in the human situation, there would be no controlled ratio of virulence between primary versus secondary virus and exposure would probably be heterosexual, via the male or female genital mucosa. The above-mentioned differences from the human situation are inevitable limitations of the animal models that make direct extrapolation and predictions of risk or prevalence in humans uncertain. However, our results demonstrate clearly that, in contrast to ‘superinfections’, secondary infections do occur more frequently than expected in controlled experimental settings. Indeed, they confirm that, despite apparent control of virus load by adaptive immune responses established after primary infection, subsequent infections can take place readily (Altfeld et al., 2002) and possibly at a higher frequency than expected. If true for the human situation, these two observations may have important implications. Secondary infections may be suppressed successfully by the host immune response and remain undetected at low levels indefinitely. Alternatively, other events with immunological consequences may result in new variants emerging that are capable of escaping specific current effector responses. Such viruses may eventually reach sufficiently high concentrations in blood or mucosal secretions to be transmitted.

Clearly, cases of secondary infection, as well as so-called superinfection, may eventually lead to intersubtype recombination (Fang et al., 2004). This concern is further underscored by recent reports on the increasing prevalence of recombinant forms of HIV-1 in certain populations (Carrion et al., 2003; Cornelissen et al., 2000; Koulinska et al., 2001; Laukkanen et al., 2000; Montavon et al., 1999; Peeters & Sharp, 2000; Peeters et al., 2003; Yang et al., 2002; Yu et al., 2001). Already faced with the problems of antigenic drift due to the high mutation rate, the additional complexity of possible antigenic shift due to the emergence of complex HIV-1 chimeras may become an even greater impediment for the generation of prophylactic vaccines (Barouch & Letvin, 2002; Domingo et al., 1997; Heeney & Hahn, 2000; Klenerman et al., 2002).

Finally, in addition to the possible public-health implications, our findings stress the importance of stringent criteria for the virological and longitudinal follow-up of at-risk individuals, even if they are compliant with antiretroviral therapy. Secondary infections and superinfections can be difficult to detect in the clinic, unlike experimental preclinical settings. PCR assays using generic primers may detect only the most predominant strain, whereas a choice of specific primers can only be made if the sequences of the possible infecting strains are known, which is rarely the case in the clinic. With the real threat of new HIV-1 recombinants arising in the AIDS epidemic, detailed monitoring and cautious counselling of infected persons are warranted.


   ACKNOWLEDGEMENTS
 
The authors thank Lucy Verkade, Henk van Westbroek and David Davis for their assistance in the preparation of this manuscript and the personnel responsible for the animal facilities. This work was supported by EC grants QLK2-CT-1999-00871, BMH4-CT97-2145, BMH4-CT97-2067 and QLK2-CT-1999-01321 and NIH grant 1P01-AI48225-01A2.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 23 April 2004; accepted 12 September 2004.



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