Swedish Institute for Infectious Disease Control and Microbiology and Tumor Biology Center, Karolinska Institute, SE-171 82 Solna, Sweden1
Institute for Molecular Virology, GSF National Research Centre for Environment and Health, Trogerstr. 4b, 81675 Munich, Germany2
Department of Virology, Biomedical Primate Research Centre, 2280 GH Rijswijk, The Netherlands3
Department of Virology, The National Veterinary Institute, Uppsala, Sweden4
Author for correspondence: Rigmor Thorstensson. Fax +46 8 337460. e-mail rigmor.thorstensson{at}smi.ki.se
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Abstract |
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Introduction |
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In the development of an AIDS vaccine, no single immune correlate to protective immunity has been defined (Heeney et al., 1999 ). In the absence of a single immune response correlate that will predict vaccine efficacy, non-human primate models are important. Macaques infected with simian immunodeficiency viruses (SIVmac, SIVsm and SIVmne) develop a disease similar to AIDS. By using this model, induction of vaccine protection against both infection and disease can be assessed (Bogers et al., 2000
).
The best protection against SIV has been elicited by live attenuated vaccines (Putkonen, 1996 ). However, live attenuated, multiply deleted SIV has been shown to cause AIDS in infant and adult macaques (Baba et al., 1999
). Furthermore, prolonged infection with a nef-defective HIV-1 strain that showed characteristics similar to attenuated SIV vaccines caused AIDS in individuals who previously showed no sign of disease progression (Learmont et al., 1999
; Rhodes et al., 2000
). Thus, attenuated HIV vaccines may not be applicable to man because of issues of safety.
Primeboost regimens induce both humoral and cellular immune responses and are therefore attractive alternatives. Combinations of virus vectors and proteins, naked DNA and proteins, naked DNA and virus vectors and combinations of different virus vectors have been tested (Excler & Plotkin, 1997 ; Barnett et al., 1998
; Heeney et al., 2000
).
Modified vaccinia virus Ankara (MVA) is a highly attenuated poxvirus vector under investigation as a vector for possible HIV vaccine candidates. Although MVA is replication-deficient in human cells, it expresses recombinant proteins at levels equal to that of fully replication-competent vaccinia virus (Sutter & Moss, 1992 ). Recombinant MVASIV has been shown to induce neutralizing antibodies, T-cell proliferative responses and CTL responses in macaques. Furthermore, partial protection has been reported against intravenous homologous SIV challenge (Hirsch et al., 1996
; Ourmanov et al., 2000
; Seth et al., 2000
).
Immune-stimulating complexes (ISCOMs) are lipid particles that comprise the immunostimulatory fractions from Quillaia saponaria (Quil A), cholesterol and phospholipids (Barr et al., 1998 ). Immunization of HIV envelope glycoproteins incorporated into ISCOMs has been shown to induce antibodies, T-cell proliferation (Verschoor et al., 1999
; Nilsson et al., 1995
) and CTLs (Heeney et al., 1994
). Furthermore, we reported long-term protection against intravenous HIV-2 challenge in macaques induced by immunization with native HIV-2 Env in ISCOMs or Ribi adjuvant (Nilsson et al., 1995
).
Early HIV-1 vaccine studies focused on Env as the immunogen. Lately, primeboost vaccination regimens have been initiated using vaccinia virus vector priming and protein boosts (Mulligan & Weber, 1999 ). In the SIVmne/Macaca nemestrina model, Polacino et al. (1999b
) reported complete protection against intravenous infection with pathogenic, uncloned SIVmne by vaccinia virus priming and multiple protein-boost immunizations in Freunds incomplete adjuvant that included both envelope and core antigens.
In the present study, we evaluated the immunogenicity and protective efficacy against intrarectal SIV challenge of an MVASIVsm vaccine and a primeboost regimen in which MVASIVsm was combined with a single ISCOM-formulated protein boost of gp148 and p27. A stronger humoral and cellular immune response was elicited by the primeboost regimen than by vaccination with MVASIVsm alone. After intrarectal challenge, all control monkeys as well as all but one vaccinee given the combined vaccine became infected. Prolonged survival was seen in two of four monkeys in each of the groups immunized with MVASIVsm, in two of the three infected monkeys given the combined vaccines and in three of four monkeys given MVA wild-type, compared with naive controls.
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Methods |
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Immunogens.
The recombinant virus MVASIVsm co-expresses the gagpol and env coding sequences of SIVsmmH4 (kindly provided by Bernard Moss, NIH, Bethesda, MD, USA) (Hirsch et al., 1996 ). gagpol was placed under the transcriptional control of the natural vaccinia virus earlylate promoter P7·5 and env was expressed using a strong synthetic vaccinia virus earlylate promoter (Chakrabarti et al., 1997
).
For the production of vaccines, MVASIVsm and non-recombinant MVA were amplified on primary chicken embryo fibroblasts (CEF) and purified by ultracentrifugation through a cushion of 36% sucrose. Purified viruses were reconstituted in PBS and titrated by end-point dilution in CEF to obtain the TCID50 (infectious units), aliquotted and stored at -70 °C.
The MVASIVsm vaccine preparations were controlled for synthesis of recombinant proteins by immunostaining and Western blot analysis of infected CEF cell preparations by using sera from an SIVsm-infected macaque (data not shown).
Native SIVsm envelope glycoprotein gp148 purified by affinity chromatography (Gilljam, 1993 ) and recombinant SIVmac251 p27 (rp27) were used for booster immunizations. The proteins were conjugated to ISCOM matrix (Lövgren & Morein, 1988
), consisting of a mixture of low-tox Quillaia saponin fractions QH-A and QH-C (Verschoor et al., 1999
), essentially as described by de Vries et al. (1994)
.
MVASIVsm recombinant viruses and rp27 were provided by the EU programme EVA/MRC Centralised facility for AIDS Reagents, NIBSC, UK. Purified SIVsm gp148 was prepared by the Department of Virology, Swedish Institute for Infectious Disease Control, Solna, Sweden.
Study design.
Three groups of four monkeys (n=12) were inoculated intramuscularly (i.m.) with 5x108 p.f.u. each of MVASIVsm env and gagpol, one group (A) at 0 and 3 months, another group (B) at 0, 3 and 8 months and a third group (C) at 0 and 3 months followed by 50 µg purified native SIVsm gp148 and 50 µg recombinant SIVmac p27 in ISCOMs at 8 months. One month after the last immunization, the vaccinees, together with four naive control monkeys (group D) and four monkeys immunized with wild-type MVA at 0, 3 and 8 months (group E), were challenged intrarectally with 10 MID50 cell-free, monkey-cell-grown SIVsm. On the day of challenge, aliquots of liquid-nitrogen-stored SIVsm were thawed, pooled and diluted 1:10. Three ml of the virus dilution was delivered intrarectally atraumatically to each monkey using a paediatric feeding tube (Quesada-Rolander et al., 1996 ).
The monkeys were monitored for clinical changes and blood samples were collected for immunological and virological studies. The study was completed 27 months after challenge (groups BE) or 32 months after challenge (group A).
Detection of antibody responses.
ELISAs were used to detect antibodies in serum to native SIVsm gp148 as described in detail previously (Nilsson et al., 1995 ). To detect antibodies to p27, the antigen was changed to rp27 diluted to 0·5 µg/ml for coating. The ELISA titres were defined as the reciprocal of the highest serum dilution that gave an absorbance value more than twice that of individual pre-immunization sera.
The presence of antibodies mediating SIVsm-neutralizing activity was determined in an assay using PHA-stimulated human PBMCs (Zhang et al., 1997 ) and an SIVsm stock virus that had been grown on monkey PBMCs. Neutralizing-antibody titres were defined as the dilution of serum that resulted in at least 90% reduction in antigen synthesis, compared with control wells that did not contain serum, by using an in-house antigen ELISA (Thorstensson et al., 1991
)
SIV Env-specific IgA antibodies in serum and rectal washes were determined in an ELISA using SIVsm gp148 diluted to 1 µg/ml for coating, as described in detail elsewhere (Nilsson et al., 2001 ). A sample was considered positive if the absorbance was greater than twice that of pre-immunization samples. Rectal washes were collected 9 or 14 days after the last immunization. The rectal mucosa was flushed with 2 ml PBS and aspirates were collected, centrifuged and stored at -70 °C.
T-cell proliferation assay.
T-helper cell responses were determined in a standard [3H]thymidine incorporation assay using HIVSBL6669 whole virus antigen (5 µg/ml) to stimulate proliferation as described elsewhere (Nilsson et al., 2001 ). Stimulation indices (SI) were calculated by dividing the mean c.p.m. of the antigen-stimulated wells by the mean of the unstimulated wells. An SI
2·0 was considered positive.
CTL assay.
The cytolytic activity of T lymphocytes was determined as described previously (Mäkitalo et al., 2000 ; Andersson et al., 1996
). Target cells were an autologous herpesvirus papio-transformed B lymphoblastoid cell line (B-LCL) infected with recombinant vaccinia virus expressing SIVmac Gag/Pol or Env products or with vaccinia virus only as control. One part 51Cr-labelled target cells was mixed with 40 parts unlabelled, MVA-infected target cells (cold targets) to reduce vaccinia virus-specific cytolysis. For generation of effector cells, monkey PBMCs were stimulated with concanavalin A (Sigma) and cultured for 1421 days in medium containing IL-2 (Amersham). An effector-to-target cell ratio of 100:1 was used. Specific lysis (%) was calculated by the following formula: 100x(experimental release-spontaneous release)/(maximum release-spontaneous release). A specific chromium release of >4% was considered positive for SIV Gag/Pol CTL (Nilsson et al., 2001
). The criterion for a positive Env CTL was based on results with or without CD8+ T-cell depletion. Thus, Env CTL was considered positive when the difference in chromium release was >10%. However, the trend for each animal was always considered and a single positive value was not accepted unless confirmed on another occasion.
CD8+ T lymphocytes were enriched or depleted by using immunomagnetic beads (Dynabeads; Dynal) according to the manufacturers instructions.
Assessment of virus infection after challenge.
Virus re-isolation was performed essentially as described previously (Nilsson et al., 1995 ) using 2x106 macaque PBMCs and 1·8x107 human PHA-stimulated PBMCs for co-cultivation. In order to detect SIV proviral DNA in lymphocytes, a nested PCR and primers specific for the long terminal repeat (LTR) and gag genes were used (Walther et al., 1998
). SIV RNA levels in plasma were assessed by using a highly sensitive quantitative competitive (QC) RTPCR assay with a detection limit of 40 RNA equivalents/ml plasma (ten Haaft et al., 1998
).
Lymphocyte subset analysis.
Immunized monkeys were monitored for changes in their T-lymphocyte subsets by flow cytometry analysis, as described previously (Mäkitalo et al., 2000 ). Briefly, EDTA-treated whole blood was stained with fluorescein-conjugated anti-CD8 and phycoerythrin-conjugated anti-CD4 antibodies. After lysis of the red blood cells and fixation, quantification of cell-surface immunofluorescence was done in a FACScan flow cytometer and analysis was performed using CELLQuest software (Becton-Dickinson Immunocytometry Systems).
Statistical analysis.
Statistical calculations were made using the MannWhitney U-test for non-parametric observations.
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Results |
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All vaccinees had undetectable anti-Env antibody levels after the first MVASIVsm immunization. One month after the second MVASIVsm immunization, low anti-Env antibody titres (range 100500) were detected in 9 of 12 vaccinees (Fig. 1ac
). Furthermore, 14 days after the third MVASIVsm immunization, given to monkeys in group B, a 25-fold increase in anti-Env antibody titre was seen in all vaccinees (Fig. 1b
). A larger, more than 600-fold increase in anti-Env antibody titre was detected in all primeboosted vaccinees (group C) 14 days after the Env and Gag protein boost (Fig. 1c
).
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Sera collected 9 or 14 days after the last immunization were analysed for anti-SIV gp148 IgA antibodies. Borderline levels of anti-Env IgA antibodies were seen in 3 of 4 monkeys immunized twice with MVASIVsm. Higher levels were seen in monkeys immunized three times with MVASIVsm and even higher levels were seen in primeboosted vaccinees. In parallel, anti-Env IgA antibodies were also detected in rectal washes from all monkeys immunized twice with MVASIVsm, in 2 of 4 monkeys immunized three times with MVASIVsm and in 2 of 4 primeboosted vaccinees (Table 1).
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Immune responses at the time of challenge
The immune responses at the time of challenge are summarized in Table 1. At this time-point, 1 month after the last immunization, we confirmed the trend seen shortly after the final immunization, such that, when anti-Env antibodies exhibiting binding or neutralizing activity and T-cell proliferative responses were studied, clear differences were seen between the three groups of immunized monkeys. Monkeys given two MVASIVsm immunizations followed by a protein boost had strong to moderately strong immune responses, monkeys given three immunizations with MVASIVsm had weak immune responses and monkeys given two immunizations with MVASIVsm had weak immune responses or levels of immune responses that were below the detection limit. Furthermore, titres of antibodies to SIV p27 and SIV Gag/Pol-specific CTLs were unchanged and remained low or undetectable.
Outcome of intrarectal SIVsm challenge
One month after the last immunization, the vaccinees (groups AD) and four naive controls were challenged intrarectally with 10 MID50 SIVsm. One monkey, D19, immunized twice with MVASIVsm and boosted with gp148 and p27 in ISCOMs, was repeatedly virus isolation-negative, lacked proviral DNA in PBMC and exhibited a reduction in circulating antibodies (data not shown and Fig. 1). No virus RNA could be detected in plasma after challenge by using a highly sensitive QC RTPCR assay (Fig. 3
). The monkey was considered to be protected completely. All the other vaccinees, as well as the control monkeys, became infected. Varying degrees of virus isolation positivity over time and varying levels of virus RNA in plasma were displayed (Fig. 3
). Virus load data obtained 3 months after challenge were compared because viraemia levels at this time-point had previously been suggested to be predictive of disease outcome. ten Haaft et al. (1998)
reported that a virus load that remained below a threshold of 104 RNA equivalents/ml plasma indicated a non-pathogenic course of infection. Monkeys D9 and D11, both immunized three times with MVASIVsm, displayed a reduced frequency of positive virus isolation and a low virus copy number. Monkey D2, immunized twice with MVASIVsm, displayed an even lower frequency of virus isolation positivity and a plasma RNA load of 40 copies/ml plasma. Another monkey in this group (D3) yielded virus at 15 of 20 time-points and had a plasma copy number of 5x103 3 months after challenge. Also, three of four monkeys immunized three times with wild-type MVA displayed a reduced frequency of positive virus isolation (D5, D6 and D7) and their plasma RNA copy numbers 3 months after challenge ranged between 103 and 5x103. The remaining vaccinees, two monkeys immunized twice with MVASIVsm (group A), two monkeys immunized three times with MVASIVsm (group B), three primeboosted monkeys (group C) and one monkey immunized three times with wild-type MVA (group D), were generally virus isolation-positive on every occasion tested and most yielded virus copy numbers of more than 5x104 copies/ml plasma 3 months after challenge. Even higher virus copy numbers (>105) were detected in three of four of the naive control monkeys (D22, D23, D24). The other naive control monkey (D21) exhibited a lower virus copy number, with 103 viral RNA copies/ml plasma at this time-point. The virus load at 2 weeks after challenge was significantly lower in the SIV vaccinees compared with the naive and wild-type MVA-vaccinated controls (P=0·0008; MannWhitney U-test). However, no statistically significant difference was seen when comparing the virus loads at 3 months.
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All vaccinees immunized three times with MVASIVsm showed anamnestic antibody responses to SIV Env, as determined by ELISA. However, only monkeys D9 and D10 yielded antibodies exhibiting neutralizing activity (titre of 40) 14 days after challenge. Among the monkeys immunized twice with MVASIVsm and boosted with protein, the protected monkey (D19) showed a steady drop in anti-SIV Env antibodies (Fig. 1). Monkeys D17 and D18 maintained a constant anti-Env antibody titre of 62500 until 9 months after challenge, when a 5-fold increase in antibody titre was seen (data not shown). Fourteen days after challenge, monkey D18 exhibited high neutralizing-antibody responses, yielding a titre of 640. The fourth monkey in this group (D20) exhibited a strong anamnestic antibody response of binding antibodies (Fig. 1
). However, only a 2-fold increase in neutralizing-antibody titre was detected.
All MVA wild-type controls had seroconverted by 1 month after challenge (Fig. 1). Anti-SIV Env antibodies detected by ELISA did not reach levels comparable to those in the other vaccinees until 2 or 3 months after infection. However, SIVsm-neutralizing antibody titres in monkeys D6 and D7 were similar to those seen in the MVASIVsm-vaccinated monkeys by 1 month after infection (titre range 80640).
Cell-mediated immune responses after intrarectal SIVsm challenge
A strong T-cell proliferative response to HIV-2SBL6669 was observed in monkey D19, the monkey that was protected from SIV infection, for more than 8 months after challenge. In contrast, all infected vaccinees lost lymphoproliferative responses after challenge. Furthermore, monkeys D5 and D7, which had received wild-type MVA vaccination, displayed strong T-cell proliferative responses 4 months after challenge (Fig. 2).
SIV Env-specific CTLs were regularly detected during the first year of follow-up in two vaccinees. Cytolysis ranged between 1117 and 1423% for two primeboosted monkeys (D17 and D18). In one monkey immunized three times with MVASIVsm (D10), cytolytic activities of 26, 16 and 16% were detected at 3, 9 and 12 months after challenge. Monkey D16, immunized twice with MVASIVsm, had detectable SIV Gag/Pol-specific cytotoxic activities of 15 and 16% at 1 and 2 months after challenge. None of the other seven SIV vaccinees tested showed any cytolytic activity at any of the five time-points between 1 and 12 months after challenge. Furthermore, none of the wild-type MVA-immunized monkeys developed detectable CTLs in peripheral blood (data not shown).
Clinical outcome of intrarectal SIVsm challenge
The monkeys were monitored for changes in CD4+ T-cell counts, haematology, body weight and signs of disease over a period of more than 2 years after challenge. Fig. 4 summarizes the CD4+ T-cell counts and survival time in the vaccinees and control monkeys.
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Two infected monkeys, immunized two (D3) or three (D9) times with MVASIVsm, showed little change in their CD4+ T-cell counts and they remained healthy during the study period. Yet another monkey (D11), immunized three times with MVASIVsm, showed a moderate decline in CD4+ T cells and it also stayed healthy during the follow-up. Surprisingly, three of four wild-type MVA-immunized monkeys (D5, D6 and D7) remained healthy, with normal CD4+ T-cell counts. Moreover, although the fourth wild-type MVA-immunized monkey (D4) showed a progressive CD4+ T-cell depletion after challenge and, by 11 months, had lost most of its CD4+ T cells, it was not euthanized until 15 months later, when it started to lose weight.
The other three monkeys immunized twice with MVASIVsm developed clinical signs of disease within 32 months after challenge. Monkey D1 showed a slow but steady decline in CD4+ T cells, which dropped rapidly after 26 months of infection. It was euthanized 4 months later. Monkey D16 was euthanized 21 months after challenge, together with monkey D2, which, despite an initially low level of plasma virus RNA, showed a severe depletion of CD4+ T cells beginning 1 year after challenge.
Two monkeys, D8 and D10, that had received MVASIVsm three times, showed progressive declines in CD4+ T cells and D10 was euthanized 16 months after challenge. Although D8, at that time-point, had an equally low CD4+ T-cell count, it was not euthanized until 10 months later, when it had started to lose weight.
Among the monkeys immunized with MVASIVsm in combination with a protein boost, another two vaccinees (D17 and D18) showed moderate changes in CD4+ T cells during the first 16 months of follow-up. D17 was euthanized 10 months later because of clinical signs of disease. Monkey D18 remained clinically healthy during the follow-up period. The last monkey in the group (D20) lost CD4+ T cells rapidly and was euthanized 10 months after challenge.
The rate of disease progression in monkey D20 was similar to that observed in the SIVsm-infected control monkeys. All these controls exhibited rapid CD4+ T-cell losses. A near total depletion was already observed after 6 months of infection in monkey D24, which was euthanized 6 months later. The other controls were euthanized within 18 months of infection.
The survival time of the monkeys immunized with wild-type MVA was prolonged significantly compared with that of 26 naive historic controls challenged intrarectally with SIVsm (P=0·043, MannWhitney U-test).
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Discussion |
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By combining the MVASIVsm vaccine with a protein boost, strong T-helper cell responses and high neutralizing-antibody titres were induced. The T-cell proliferative responses detected were similar to those reported in human trials after two immunizations with ALVACHIV-1 (vCP125) and boosting with rgp160 in alum or Freunds incomplete adjuvant (Pialoux et al., 1995 ). Both T-helper cell responses and neutralizing-antibody responses were stronger than those we reported previously after giving two immunizations with ALVAC expressing HIV-2 Env, Gag and Pol and a boost of native HIV-2 gp125 in QS21 adjuvant (Andersson et al., 1996
). The stronger neutralizing-antibody response could be attributed in part to the use of a more sensitive assay, using virus grown on monkey PBMCs. The T-cell proliferative responses also exceeded those seen after immunizations with either SIV Env formulated in ISCOMs or DNA priming and boosting with rgp120 in ISCOMs (Verschoor et al., 1999
).
A potential HIV vaccine candidate will have to protect against mucosal challenge. We therefore challenged monkeys intrarectally with the homologous SIVsm. In order to ensure that all monkeys were exposed to the same virus dose, 3 ml pooled and diluted virus was delivered intrarectally to each monkey. After challenge, all naive control monkeys, as well as all but one of the vaccinees given the MVASIV primeprotein boost, became infected. Control of viraemia was seen in two of four monkeys in each of the groups immunized with MVASIVsm, in two of the three infected monkeys given the combined vaccines and in three of four monkeys given wild-type MVA. Previous studies have shown partial protection against AIDS after intravenous challenge with a highly related SIVsm virus strain after MVASIVsmH-4 vaccination with (Hirsch et al., 1996 ) or without (Ourmanov et al., 2000
) boosting with whole inactivated SIV. In M. nemestrina, Polacino et al. (1999b)
reported complete protection against intravenous infection with pathogenic, uncloned SIVmne by vaccinia virus primeprotein boost immunizations including both envelope and core antigens. Protection against intrarectal challenge was reported after priming with recombinant vaccinia virus expressing SIVmne envelope gp160 and multiple immunizations with gp160 in incomplete Freunds adjuvant (Polacino et al., 1999a
). Moreover, after four immunizations with another attenuated pox vector, NYVAC, expressing SIVK6W env, gag and pol genes, partial protection was demonstrated against intrarectal SIVmac251 challenge (Benson et al., 1998
). Thus, the present study supports and extends these findings to show that MVASIVsm in combination with a single protein boost in ISCOMs may prevent infection or delay immunosuppression and disease caused by mucosal SIV infection.
Following the intrarectal SIVsm challenge, given 1 month after the final immunization, three of four monkeys immunized with 5x108 p.f.u. wild-type MVA (no SIV gene) exhibited control of viraemia and remained clinically healthy. All wild-type MVA-immunized monkeys initially exhibited high virus loads. By 3 months after challenge, viraemia had dropped to a plasma virus copy number of between 103 and 5x103 in three of the monkeys (D5, D6 and D7). These three monkeys also exhibited low virus-isolation frequencies and they maintained normal CD4+ T-cell levels during the follow-up. Hanke et al. (1999) have reported protection in a wild-type MVA-immunized rhesus macaque challenged intrarectally with SIVmac 2 months after the last MVA immunization. However, when wild-type MVA vaccinees were challenged intrarectally 3 months after the last immunization, no protective effect was seen (Nilsson et al., 2001
). Although not fully proven, these data suggest that MVA may induce a short-lived, non-specific antiviral activity. In the present study, we found that the control of viraemia and maintenance of CD4+ T cells was associated with persistent T-cell proliferative responses (Fig. 2
). Blanchard et al. (1998)
have reported that MVA is a potent inducer of IFN type I. Furthermore, it has been shown that, unlike other vaccinia virus strains, MVA does not express soluble receptors for IFN-
, IFN-
/
, tumour necrosis factor and CC chemokines (Antoine et al., 1998
), antiviral factors that could potentially contribute to the observed effect. Importantly, our findings stress the need for longer waiting periods after the final MVA immunization in order to allow investigations of virus-specific immune correlates of protective immunity in MVA vaccine studies. Therefore, we did not attempt to define such correlates in our study, despite the reduced viraemia seen in some of the vaccinees that received MVASIVsm two (group A) or three times (group B).
A third group (C) of monkeys was given two immunizations with MVASIVsm at 0 and 3 months and a boost of gp148 and p27 in ISCOMs at 8 months, i.e. challenged 6 months after the last MVA immunization. At this time-point, the non-specific antiviral activity of MVA should not have influenced the challenge experiment. It is interesting to note the high neutralizing-antibody titres and levels of T-cell proliferative response seen in the primeboosted vaccinees that displayed control of infection. One vaccinee (D19) was repeatedly virus isolation-negative and proviral DNA-negative in PBMCs and no virus RNA was detected after challenge by using a highly sensitive QC RTPCR assay (ten Haaft et al., 1998 ). Furthermore, D19 lacked an anamnestic antibody response in serum and retained normal CD4+ T-cell levels during more than 2 years of follow-up. The monkey was considered completely protected. At the time of challenge, monkey D19 had a high T-cell proliferative response but showed neither detectable SIV-specific CTLs nor neutralizing antibodies. After SIVsm challenge, the high T-cell proliferative responses were maintained for more than 8 months, although levels of circulating antibodies slowly waned. In addition, two other monkeys (D17 and D18) in this group showed signs of controlled infection, as judged by the virus load at 6 months after challenge and a delayed decrease in CD4+ T cells. Monkey D18 had very high neutralizing-antibody titres and levels of T-cell proliferative response at the time of challenge. Monkey D17 also had a high neutralizing-antibody titre and T-cell proliferative responses. It also had a detectable SIV Gag/Pol-specific CTL response. In contrast, the fourth monkey (D20) in this group exhibited weaker immune responses at the time of challenge and rapidly lost its CD4+ T cells. The presence of potent T cells and antibodies exhibiting virus neutralization at the time of challenge may have contributed to the balance established between the virus and the host.
We conclude that the MVASIVsm primeprotein boost protocol elicited a stronger immune response than MVASIVsm alone. Furthermore, one MVA-primed protein-boosted monkey was protected completely and another two monkeys in this group showed controlled viraemia. Immunization with MVASIVsm as well as wild-type MVA seemed to delay disease progression in a proportion of the monkeys.
Currently, clinical vaccine trials are being planned or are ongoing in which a live recombinant vector such as MVA (D. L. Birx, personal communication) or ALVAC (Excler & Plotkin, 1997 ) is combined with a subunit boost (gp120). Our findings bring hope that an MVA primeprotein boost vaccine strategy may induce immune responses potent enough to establish complete protection against mucosal HIV-1 infection or induce control of viraemia and delay disease progression.
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Acknowledgments |
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Footnotes |
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c Present address: Medical Products Agency, Box 26, 75103 Uppsala, Sweden.
d Present address: Gustavsbergs Vrdcentral, Skärg
rdsvägen 7, 134 30 Gustavsberg, Sweden.
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References |
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Received 5 September 2001;
accepted 7 December 2001.