Cross-protection against mucosal simian immunodeficiency virus (SIVsm) challenge in human immunodeficiency virus type 2-vaccinated cynomolgus monkeys

Lilian Walther-Jallow1, Charlotta Nilsson1, Johan Söderlund2, Peter ten Haaft3, Barbro Mäkitalo1, Peter Biberfeld2, Per Böttiger1, Jonathan Heeney3, Gunnel Biberfeld1 and Rigmor Thorstensson1

Swedish Institute for Infectious Disease Control and Microbiology and Tumour Biology Centre, Karolinska Institute, SE-17182 Solna, Sweden1
Immunopathology Laboratory, Karolinska Institute, SE-10401 Stockholm, Sweden2
Department of Virology, Biomedical Primate Research Centre, 2280 GH Rijswijk, The Netherlands3

Author for correspondence: Lilian Walther-Jallow at Department of Immunology, Swedish Institute for Infectious Disease Control, SE-17182 Solna, Sweden. Fax +46 8 337460. e-mail Lilian.Walther.Jallow{at}mtc.ki.se


   Abstract
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Abstract
Introduction
Methods
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Discussion
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In this study we compared the efficacy of live attenuated human immunodeficiency virus type 2 (HIV-2) vaccine alone versus boosting with live non-pathogenic HIV-2 following priming with ALVAC HIV-2 (recombinant canarypox virus expressing HIV-2 env, gag and pol). Six monkeys were first inoculated intravenously with live HIV-2SBL-6669 and 7 to 10 months later were challenged intrarectally with 10 MID50 of cell-free simian immunodeficiency virus (SIV) strain SIVsm. One monkey was completely protected against SIV infection and all five monkeys that became SIV-infected showed a lower virus replication and an initial lower virus load as compared with a parallel group of six control animals. In another experiment five monkeys were immunized either three times with ALVAC HIV-2 alone or twice with ALVAC HIV-2 and once with purified native HIV-2 gp125. The monkeys were then challenged with HIV-2 given intravenously and finally with pathogenic SIVsm given intrarectally. After challenge with SIVsm, three of five monkeys were completely protected against SIVsm infection whereas the remaining two macaques became SIV-infected but with limited virus replication. In conclusion, vaccination with an ALVAC HIV-2 vaccine followed by exposure to live HIV-2 could induce cross-protection against mucosal infection with SIVsm and seemed to be more efficient than immunization with a live HIV-2 vaccine only.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
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More than 90% of new infections with human immunodeficiency virus (HIV) occur in developing countries, where antiviral treatment has limited availability. Most infections are transmitted by sexual contact and therefore development of prophylactic vaccines that can protect against mucosal exposure is needed.

At present the macaque model is the most useful non-human primate model for testing the efficacy of AIDS vaccines. HIV-2 and the closely related simian immunodeficiency virus (SIV) strains SIVsm, SIVmac and SIVmne persistently infect cynomolgus (Macaca fascicularis) (Putkonen et al., 1989a , b ), rhesus (M. mulatta) (Daniel et al., 1985 ) and pig-tailed (M. nemestrina) (Benveniste et al., 1988 ) macaques. SIVsm, SIVmac and SIVmne cause loss of CD4+ T-cells and immunodeficiency syndromes in macaques (Daniel et al., 1985 ; Kuller et al., 1990 ; Putkonen et al., 1992 ), whereas only some strains of HIV-2 cause simian AIDS (Looney et al., 1998 ).

Live attenuated vaccines have been shown to induce the best resistance to superinfection with virulent virus in the SIV macaque model. We and others have shown that live attenuated SIV strains can confer protection against pathogenic SIV strains inoculated intravenously as well as by mucosal routes. Different clones of SIVmac have been shown to induce protection against intravenous challenge with wild-type cell-free SIVmac (Marthas et al., 1990 ; Daniel et al., 1992 ; Wyand et al., 1996 ) or with SIV-infected cells (Almond et al., 1995 ) as well as against intrarectal challenge with cell-free SIVsm (Nilsson et al., 1998 ). However, no protection was seen against the more divergent HIV-2SBL-6669 (Nilsson et al., 1998 ). Furthermore attenuated SIV clones were shown to protect against intravenous (Bogers et al., 1995 ) and intrarectal (Cranage et al., 1997 ) challenge with an SIV/HIV-1 chimera (SHIV-4). We and others have also demonstrated that non-pathogenic SHIV chimeras used as vaccine can protect from SIV superinfection following intravenous (Letvin et al., 1995 ), intrarectal (Quesada-Rolander et al., 1996 ) as well as intravaginal (Miller et al., 1997 ) challenge. Live attenuated vaccines should not be considered as candidate vaccines against human AIDS due to safety concerns, but they represent important tools to elucidate the nature of protective immune responses.

Replicating vectors such as recombinant poxvirus vectors carrying the genes encoding virus antigens have the potential to mimic virus infections and to induce both humoral and cell-mediated immunity. In the macaque model we and others have shown protection from homologous HIV-2 infection in animals immunized multiple times with HIV-2 recombinant canarypox virus or HIV-2 recombinant attenuated vaccinia virus and boosted with HIV-2 Env or V3 peptides (Andersson et al., 1996 ; Franchini et al., 1995 ). In other experiments using recombinant poxvirus expressing various SIV antigens sterilizing immunity against pathogenic SIV was not achieved but a reduction of virus load and protection from SIV-induced disease was observed in a proportion of the monkeys (Hirsch et al., 1996 ; Abimuku et al., 1997 ; Benson et al., 1998 ).

HIV-2 strain SBL-6669 (Putkonen et al., 1989a ) is non-pathogenic in cynomolgus macaques as assessed by the persistence of normal levels of CD4+ T-cells and by the lack of disease progression in animals chronically infected for up to 8 years (our unpublished observation). Thus HIV-2SBL-6669 has features in common with live attenuated vaccines.

We have previously reported that infection with HIV-2SBL-6669 does not induce sterilizing immunity against pathogenic SIVsm inoculated intravenously, but can confer protection from SIV-induced disease in cynomolgus macaques (Putkonen et al., 1990 , 1995 ). In this study we investigated the protective efficacy of HIV-2SBL-6669 alone or in combination with HIV-2 recombinant canarypox against SIVsm intrarectal challenge since prevention of mucosal transmission is an ultimate goal of a future HIV vaccine.


   Methods
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Introduction
Methods
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{blacksquare} Animals.
Seventeen purpose bred, adult cynomolgus monkeys (M. fascicularis) of both sexes were used in this study. The handling of the animals was done according to the guidelines of the Swedish Ethical Committee for Animal Protection. Prior to vaccination the animals were examined and found to be clinically healthy. The monkeys were virus isolation-negative and seronegative for simian T-lymphotropic virus, HIV-2 and SIV and also found by PCR to be negative for simian retrovirus subtype D (Liska et al., 1997 ).

{blacksquare} Vaccination and challenge.
Eleven macaques were selected from two previous vaccine experiments in which they had become HIV-2-infected either as naive control animals or as vaccinees (Andersson et al., 1996 ; our unpublished data). For a detailed immunization schedule see Fig. 1. In group I six monkeys had been intravenously inoculated with 30 MID50 (50% monkey infectious doses) of live HIV-2SBL-6669 (Fig. 1; Putkonen et al., 1991 ). The monkeys in group II were either vaccinated three times with ALVAC HIV-2 (recombinant canarypox virus expressing HIV-2 env, gag and pol genes) kindly provided by Virogenetics Corp (Troy, New York, USA) or twice with ALVAC HIV-2 and once with purified native HIV-2 gp125 (Gilljam et al., 1993 ) given in QS21 adjuvant over a period of 7 months (Fig. 1; Andersson et al., 1996 ). These five monkeys were challenged intravenously with 30 MID50 of HIV-2 1 month after the last immunization and became HIV-2-infected. Seven to 10 months later all monkeys from both groups and six naive control animals were intrarectally challenged with 10 MID50 of cell-free SIVsm (Quesada-Rolander et al., 1996 ).



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Fig. 1. Immunization schedule: one group (I) of six monkeys was intravenously inoculated with live HIV-2SBL-6669. Another group of monkeys (II) was either vaccinated three times with ALVAC HIV-2 (recombinant canarypox virus expressing HIV-2 env, gag and pol genes) or twice with ALVAC HIV-2 and once with purified native HIV-2 gp125 given over a period of 7 months. The five monkeys in group II were challenged intravenously with live HIV-2 1 month after the last immunization and became HIV-2-infected. Seven to 10 months later all 11 monkeys together with six naive control animals were intrarectally challenged with cell-free SIVsm.

 
{blacksquare} Direct detection of virus.
Virus isolation was performed by cocultivation of 2x106 monkey peripheral blood mononuclear cells (PBMCs) or lymph node cells with 18x106 human PBMCs stimulated with phytohaemagglutinin (Difco) (Nilsson et al., 1995 ). Cultures were tested for the presence of viral antigen by an HIV-2/SIV antigen capture ELISA (Thorstensson et al., 1991 ). Nested PCR, using primer systems in the LTR and vif regions, designed to discriminate between HIV-2ISY and SIVSMMH4, respectively, was performed to discriminate between HIV-2 and SIV infection in the monkeys (Walther et al., 1996 ).

SIV RNA levels in plasma were measured for the monkeys of group I by a highly sensitive quantitative competitive (QC) RT–PCR assay (ten Haaft et al., 1998 ). Virus load was monitored in samples collected at 2 weeks, 1, 2, 3 and 6 months after SIV challenge and thereafter every 6 months during the respective survival time. This recently published method uses an internal standard RNA that is coamplified in the PCR reaction. The primer and probe region extends over 267 bp in the SIV gag genome where the internal standard probe is substituted by a rearranged 26 bp region (ten Haaft et al., 1998 ). The PCR is run using 5'-biotinylated primers and detection of amplified fragments is done in ELISA format using a streptavidin–horseradish peroxidase-mediated reaction system and with SIV and internal standard probes in separate wells. The lower detection limit of the assay is 40 RNA equivalents/ml plasma.

{blacksquare} Clinical follow-up.
The monkeys were monitored daily for general clinical status. Body weight, lymph node size and haematological parameters were investigated at the time of each bleeding. The animals were monitored for changes in their T-lymphocyte subsets using flow cytometry analysis. PBMCs were stained with phycoerythrin-conjugated monoclonal antibodies to CD4+ cells (Becton Dickinson Immuno Cytometry Systems) and fluorescein isothiocyanate-conjugated monoclonal antibodies to CD8+ cells (Becton Dickinson) and analysed using the Cell Quest program (Becton Dickinson).

Histopathological changes were evaluated and graded as previously described (Biberfeld et al., 1986 ; Öst et al., 1989 ; Feichtinger et al., 1993 ) on haematoxylin/eosin-stained sections of paraformaldehyde-fixed, paraffin-embedded lymph node biopsies taken 1 year after SIV infection and at autopsy. Immunohistochemical stainings with a non-discriminating monoclonal antibody to HIV-2/SIV Gag protein (p27) were performed as described elsewhere (Li et al., 1993 ).

The five monkeys in group II were sacrificed 12 months after SIVsm challenge and the monkeys in group I were sacrificed at 56 months after SIVsm inoculation unless otherwise stated. The control animals were kept until development of symptoms of simian AIDS and were then euthanized. PBMCs as well as specimens from spleen, lymph nodes, tonsils and, in some cases, small intestine, were collected and investigated for signs of SIV infection.

{blacksquare} Determination of immune responses.
Serum samples were analysed for titres of antibodies to native SIVsm gp148 and HIV-2 gp125 envelope glycoproteins (Nilsson et al., 1995 ). The purified envelope glycoproteins were kindly provided by the Department of Virology, Swedish Institute for Infectious Disease Control, Stockholm, Sweden.

Neutralizing antibodies against HIV-2 and SIV were determined on at least two different occasions using human PBMCs as target cells (Nilsson et al., 1995 ). The virus used in the assay had been grown in monkey PBMCs.

Lymphocyte proliferative responses against whole viral HIV-2SBL-6669 lysate were determined. The assay was run as described previously (Andersson et al., 1996 ). Results of triplicate determinations were expressed as stimulation index (SI), defined as the ratio between incorporation of [3H]thymidine in the presence of test antigen and incorporation in the presence of culture medium alone. An SI>3·0 was considered positive.

The presence of HIV-2- and SIV-specific cytotoxic T-lymphocytes (CTLs) in PBMCs was assessed in a standard chromium release assay as previously reported (Andersson et al., 1996 ). Briefly, purified blood lymphocytes were stimulated in vitro for 2–4 days with concanavalin A, then medium was changed to include 20 U/ml of recombinant human interleukin-2 and the cells were cultured for another 10 days. Autologous B-cell lines infected with either recombinant vaccinia virus expressing any of HIV-2 Gag/Pol, HIV-2 Env, SIV Gag/Pol and SIV Nef protein or wild-type vaccinia virus were used as target cells. An effector cell to target cell ratio of 100:1 was usually used. The percentage specific 51Cr release was calculated as [(experimental c.p.m.-spontaneous c.p.m.)/(maximum c.p.m.-spontaneous c.p.m.)]x100. Percentage lysis of control vaccinia virus-infected B-cell lines was subtracted to yield specific lysis. Spontaneous release of target cells without effector cells was <20% of maximal release of target cells with detergent (5% Triton X-100) in all assays. Specific lysis above 4% for SIV Gag/Pol (Nilsson et al., 1998 ) and 11·8% for SIV Nef (Mäkitalo et al., 2000 ) was considered positive based on the control values in naive monkeys. The lower limit for positive HIV-2 CTLs was 4·7% for Gag/Pol and 8·7% for Env (Andersson et al., 1996 ). However, the trend in each animal was always considered and a single positive value was never accepted unless confirmed on another occasion.

{blacksquare} Statistics.
Comparison of virus isolation frequencies in the various groups was performed by applying the {chi}2 test.


   Results
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Methods
Results
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Virological status at the time of SIVsm challenge
All 11 monkeys in the two groups of vaccinees were HIV-2-infected and had seroconverted (Table 1) at the time of SIVsm challenge. In one monkey from group I (B188), HIV-2 could only be demonstrated by PCR on two occasions at 7 and 8 months post-infection. Detailed results of virus isolations and PCR have been described by Andersson et al. (1996) . At the time of intrarectal SIVsm challenge all 11 monkeys were HIV-2 virus isolation-negative but virus was detected by PCR in four out of six monkeys in group I, which were inoculated with HIV-2 only, and in all five monkeys in group II, which were immunized with ALVAC HIV-2 prior to HIV-2 infection (Table 1).


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Table 1. Virological and immunological characterization of the animals on the day of intrarectal challenge with SIVsm

 
Immune responses at the time of SIVsm challenge
At the time of SIVsm challenge all vaccinated monkeys of group I had ELISA antibody titres to HIV-2 gp125 ranging from 12500 to 312500 and lower antibody titres (500–62500) to SIV gp148, indicating the presence of cross-reacting antibodies. The vaccinated animals of group II showed ELISA antibody titres against HIV-2 gp125 ranging from 12500 to 62500 and fivefold lower antibody titres against SIV gp148. Two animals in each group showed low titres of HIV-2 neutralizing antibodies. High SIV neutralizing antibody titres were only demonstrable in one animal (B187), while the other monkeys had low or undetectable levels.

High T-cell proliferative responses against HIV-2 virus lysate were observed in eight of the 11 vaccinated monkeys (Table 1) while T-cell proliferative responses could not be determined in three of the monkeys due to high spontaneous reactivity. SIV Gag/Pol- or SIV Nef-specific CTL activity was not demonstrable in any of the seven investigated vaccinees on the day of challenge (data not shown) but HIV-2-specific CTL responses to Gag/Pol and/or Env were observed in five out of nine monkeys investigated during the HIV-2 vaccination period (Table 1; Andersson et al., 1996 ).

Outcome of intrarectal SIVsm challenge
Group I preinoculated with HIV-2.
One of six monkeys in group I (B184) was completely protected, as determined by the lack of reactivity in SIV-specific PCR of PBMCs and cells from lymph nodes (Table 2), spleen, bone marrow and small intestine (data not shown) and by the lack of anamnestic antibody response against SIV gp148 (Fig. 2 a). Five out of six monkeys became SIV-infected as shown by discriminatory HIV-2 and SIV PCR, but they had lower virus replication than the parallel group of control monkeys, as shown by the frequency of positive virus isolations (30/108 versus 84/90, P=0·005) (Table 2). Fourteen days post-challenge virus could be isolated from all five animals but thereafter only two vaccinated monkeys (B187, B188) were consistently virus isolation-positive. Some of the positive virus isolations may reflect replication of HIV-2 rather than SIV, especially when the samples were virus isolation-positive but PCR-negative for SIV. Five out of six control animals were virus isolation-positive at all time-points, while monkey C39 was intermittently positive from 3 months after challenge. In two of the vaccinated animals (B189, B190) SIV-infected PBMCs were only detectable by PCR during the first months, while SIV DNA was demonstrable in lymph node cells for the following 2 years.


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Table 2. Virus isolation and discriminative PCR of PBMCs and lymph node cells after intrarectal challenge with SIVsm of macaques preinfected with HIV-2SBL-6669

 


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Fig. 2. ELISA antibody titres against SIV gp148 at the time of and after intrarectal SIVsm challenge of macaques preinfected with HIV-2 (a) and of macaques vaccinated with ALVAC HIV-2 and infected with HIV-2 (b).

 
Two weeks after SIV challenge all the vaccinated monkeys showed lower plasma SIV RNA levels (104·1 to 106·3) than the control monkeys (106·4 to 107·7) (Fig. 3). Over time the plasma SIV RNA levels declined to below the detection limit in all the vaccinated monkeys, at least for a period of time (Fig. 4a), whereas in the control animals the SIV RNA levels remained above the detection limit at all time-points (Figs 3 and 4b).



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Fig. 3. Early plasma virus RNA load following intrarectal challenge with SIVsm. The lower detection limit of the assay is 40 RNA copies/ml.

 



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Fig. 4. Percentage CD4+ T-cells of PBMCs (——) in relation to RNA copies/ml plasma (---) after intrarectal SIVsm challenge of (a) macaques preinfected with HIV-2 and (b) control animals.

 
The number of circulating CD4+ T-cells declined in all animals 2 weeks after SIV challenge. However, the values returned to nearly normal in the vaccinated monkeys and in two SIV-infected control animals (C43, C44), while the CD4+ T-cells continuously decreased in the other four control monkeys.

Two of the vaccinated monkeys (B187, B188; progressors) developed AIDS and were euthanized 15 and 30 months after SIV challenge, respectively, both with declining CD4+ T-cell counts and increasing virus load from onset of disease (Fig. 4a). Four years after SIV challenge, three of the five SIV-infected vaccinees and the protected monkey were still healthy, with persistent HIV-2 detectable in PBMCs as well as in lymph node cells, but with SIV virus load below the detection limit and with normal CD4+ T-cell counts (Fig. 4a). These animals were sacrificed at 56 months when they still had normal CD4 values. Analysis of histological changes in lymph node biopsies taken 1 year after SIV challenge and at autopsy showed in the three vaccinated slow progressors and in the protected monkey features corresponding to chronic, reactive lymphadenopathy. Among the six non-vaccinated control monkeys all but one animal (C44) showed progression to regressive changes of their lymph nodes (data not shown) with follicular atrophy and depletion (according to the nomenclature established for HIV infection; Biberfeld et al., 1986 ; Öst et al., 1989 ) or changes due to malignant lymphoma (C38, C39).

All six control monkeys were euthanized during a 4 year follow-up period, all with high virus loads (105-107 RNA copies/ml) and declining CD4+ T-cell counts. One control animal (C43) with stable virus load of about 104 RNA copies/ml between 3 and 24 months showed the longest survival time (4 years), while all four control animals with more than 105 SIV RNA copies/ml died between 17 and 23 months. Control animal C39 showed a rapid initial decline of plasma virus load and a low virus isolation frequency (7/13), but at 12 months a dramatic increase of viral RNA and a decline of CD4+ T-cells was observed and the animal was euthanized at 18 months due to a high-grade malignant B-cell lymphoma (Castaños-Vélez et al., 1999 ).

Group II immunized with ALVAC HIV-2 prior to HIV-2 infection.
In group II three out of five monkeys (B176, B179, B185) were completely protected against SIVsm infection, as determined by HIV-2- and SIV-specific PCR (Table 2) and by the lack of anamnestic antibody response against SIV gp148 (Fig. 2b). The other two monkeys (B182, B186) became SIVsm-infected but with a limited virus replication in comparison with the control animals, as measured by virus isolation frequency (6/24 versus 84/90, P=0·005) (Table 2) and with stable levels of CD4+ T-cells during the year of follow-up (data not shown). At autopsy 1 year after SIV exposure SIV could not be detected by PCR in PBMCs, or cells from lymph nodes, spleen and tonsils in the three protected animals, while SIV was demonstrable in PBMCs as well as in lymph nodes of the two infected monkeys.


   Discussion
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Abstract
Introduction
Methods
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Discussion
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In this study protection against intrarectal challenge with SIVsm was achieved in four of 11 macaques vaccinated with live HIV-2 alone or in combination with ALVAC HIV-2, while all six control animals became infected. In the group of animals vaccinated with HIV-2 alone protection against SIV replication was induced in one of six monkeys. Furthermore three of the five SIV-infected animals showed low virus load and restricted SIV replication and appeared to be slow progressors with stable CD4+ T-cell counts. Also, the two vaccinated monkeys defined as rapid progressors (B187, B188) showed in the acute phase of infection a lower virus load than any of the six control animals. Interestingly, the two rapid progressors showed the lowest HIV-2 replication prior to SIV challenge. Other studies have shown that the protective efficacy of live attenuated SIV vaccines is correlated to the replicative capacity of the vaccine inoculum (Wyand et al., 1996 ) and that the strength of the host immune response is inversely correlated with the degree of virus attenuation (Lohman et al., 1994 ). In a study similar to ours Wakrim et al. (1996) found complete protection in one of six animals preinfected with HIV-2 and challenged intrarectally with SIVsm but no protection against SIV infection or against immunodeficiency and disease progression in the other five animals. However, in that study at the time of SIV inoculation the only evidence of HIV-2 infection was low antibody titres (102-103). This may explain the low protective efficacy compared to that of the present study, in which all animals vaccinated with live HIV-2 exhibited high antibody levels (104·1-105·5) and most were also PCR-positive.

In agreement with our previous study (Putkonen et al., 1995 ) we found that sterilizing immunity was not a prerequisite for protection against AIDS. Controlled SIV replication, characterized by a transient low level primary plasma viraemia followed by a rapid decline in viral RNA levels to below the detection limit of quantification, was observed in the three infected animals that survived with maintained CD4+ T-cell counts during the 56 months of follow-up. A prolonged disease-free survival time associated with limited virus replication has also been observed after SIV infection of macaques immunized with SIV recombinant attenuated poxvirus such as modified vaccinia virus Ankara (Hirsch et al., 1996 ), NYVAC or ALVAC (Benson et al., 1998 ). It seems that a certain threshold of virus load 6 to 12 weeks post-infection is predictive of a pathogenic disease course not only in vaccinated monkeys but also in naive macaques infected with different virus strains (ten Haaft et al., 1998 ; Watson et al., 1997 ). In a study by ten Haaft et al. (1998) viral RNA levels above 105 copies/ml plasma were found to be associated with development of immunodeficiency and disease progression while steady state virus loads below 104 RNA copies/ml plasma were observed after infection with non-pathogenic SIV or SHIV. Between 6 and 12 weeks post-infection all SIV-infected control animals in the present study had>105 RNA copies/ml plasma while all the vaccinated monkeys had virus loads less than 104 copies/ml plasma. It is noteworthy that the QC–PCR assay used in this study has a high sensitivity, with a lower detection limit of 40 RNA copies/ml, whereas the assays used in some other SIV vaccine studies had a detection limit of 5000 RNA copies/ml plasma (Benson et al., 1998 ; Hirsch et al., 1996 ).

A difference in protective efficacy dependent on the route of exposure was not apparent in our studies in contrast to a recent vaccine trial in rhesus macaques showing that the route of infection was important for determination of vaccine efficacy (Benson et al., 1998 ). In our previous experiment (Putkonen et al., 1995 ) using heterologous intravenous SIV challenge all four animals preinfected with HIV-2 became superinfected but with a limited virus replication and a long disease-free survival time as compared to the naive control monkeys. All four control animals developed AIDS and were euthanized in less than 2 years while three of four vaccinated animals were healthy with normal CD4+ T-cell counts for more than 5 years. In the present study one of six HIV-2-preinfected animals completely resisted heterologous SIV inoculated intrarectally and three animals remained disease-free during almost 5 years of follow-up time. On the other hand two animals were unable to control the SIV replication despite a low virus load during primary infection and were euthanized with AIDS within 18 months. There may be several explanations for the seemingly conflicting results between the study of Benson et al. (1998) and our study, such as different types of vaccine, challenge viruses, macaque species and follow-up time.

In the present study the group of animals immunized with ALVAC HIV-2 prior to HIV-2 infection showed a more solid protection against heterologous SIV inoculated intrarectally. Thus three of five monkeys resisted exposure to pathogenic SIV and the other two animals showed a limited virus replication and no sign of immunodeficiency during 1 year of follow-up. This indicates that preimmunization with ALVAC HIV-2 prior to HIV-2 infection induced a more potent immunity than live HIV-2 only. In agreement with previous HIV-2 and SIV vaccine trials in macaques no obvious correlation was found between any of the immunological parameters studied and protection (reviewed in Almond & Heeney, 1998 ; Andersson et al., 1996 ; Nilsson et al., 1995 , 1998 ; Putkonen et al., 1991 ). We previously found that a high binding antibody titre to SIV gp148 was indicative of an unprotected monkey whereas a moderate SIV gp148 binding antibody titre was indicative of a protected animal (Nilsson et al., 1998 ). Indeed in the present study the two animals exhibiting the highest levels of HIV-2 gp125 and SIV gp148 antibodies were not protected. Furthermore the only three animals showing any evidence of SIV neutralizing activity became infected. Although the role for humoral immunity in protection against SIV in the macaque model is controversial, neutralizing antibodies are probably important on mucosal surfaces, in local lymph nodes and in the circulation to control cell-free viraemia. In this study we did not investigate the antibody response at the mucosal surface. However, it has been reported that seropositive SIV-infected macaques have a significant level of mucosal SIV antibodies (Kuller et al., 1998 ).

Lymphocyte proliferative responses to HIV-2 virus lysate were demonstrated in all investigated animals on the day of SIV challenge but there was no correlation between the levels of response and protection. At the time of challenge none of the animals, whether protected or not, had demonstrable HIV-2- or SIV-specific CTL responses. Two of four protected monkeys showed HIV-2-specific CTL responses during the immunization period, but also three of five non-protected animals had demonstrable CTLs prior to SIV challenge.

Although no clear correlation between the presence of cellular immune responses and protection was observed in the present study previous studies show that CD8+ T-cells play a critical role in the control of both HIV and SIV infection. CD8+ cell-mediated host defence involves the generation of virus-specific CTL responses but also production of cellular antiviral factors, i.e. {beta}-chemokines. We have previously shown that HIV-2-exposed but seronegative cynomolgus macaques, which were resistant to mucosal SIV challenge, had SIV-specific CTLs and produced high levels of CD8+ cell-dependent antiviral factors (Putkonen et al., 1997 ; Ahmed et al., 2001 ). An inverse correlation between vaccine-induced SIV-specific CTL precursor frequency and virus load after SIV challenge has been reported, suggesting a protective role of CTLs (Gallimore et al., 1995 ). Several groups have shown that high levels of {beta}-chemokines produced by CD8+ T-cells are associated with protection against infectious SIV challenge (Lehner et al., 1996 ; Wang et al., 1998 ; Ahmed et al., 1999 ).

The results of the present study together with our previous findings (Andersson et al., 1996 ) indicate that prime boost immunization including HIV-2 ALVAC has a good protective efficacy reflected by induction of sterilizing immunity and by protection against the pathogenic consequences of a heterologous challenge virus. The implication for future vaccine trials in humans is that the vaccine efficacy must be determined not only as protection against infection but also as protection against immunodeficiency.


   Acknowledgments
 
We thank Reinhold Benthin, Hélène Fredlund and Katarina Karlén for expert technical assistance. This work was supported by grants from the Swedish International Development Cooperation Agency, Department for Research Cooperation (SAREC) and the Swedish Medical Research Council.

Materials used in this study were kindly provided by Dr E. W. Rud and Dr H. C. Holmes and the MRC Directed Programme Reagent Project, Programme EVA and the EC Programme on AIDS Research.


   References
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Abstract
Introduction
Methods
Results
Discussion
References
 
Abimuku, A. G., Robert-Guroff, M., Benson, J., Tartaglia, J., Paoletti, E., Gallo, R. C., Markham, P. D. & Franchini, G. (1997). Long-term survival of SIVmac251-infected macaques previously immunised with NYVAC-SIV vaccines. Journal of Acquired Immune Deficiency Syndromes and Human Retrovirology 15, 78-85.

Ahmed, R. K. S., Nilsson, C., Wang, Y., Lehner, T., Biberfeld, G. & Thorstensson, R. (1999). {beta}-Chemokine production in macaques vaccinated with live attenuated virus correlates with protection against simian immunodeficiency virus (SIVsm) challenge. Journal of General Virology 80, 1569-1574.[Abstract]

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Received 17 July 2000; accepted 7 March 2001.



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