1 Swedish Institute for Infectious Disease Control, SE-171 82 Solna, Sweden
2 Microbiology and Tumor Biology Center, Karolinska Institute, Sweden
3 Institute of Odontology, Karolinska Institute, Huddinge, Sweden
4 Institute of Molecular Virology, GSF-National Research Center for Environment and Health, Muenchen, Germany
5 Department of Virology, Biomedical Primate Research Centre, Rijswijk, the Netherlands
Correspondence
B. Mäkitalo
barbro.makitalo{at}smi.ki.se
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ABSTRACT |
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INTRODUCTION |
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To improve the immunogenicity of DNA vaccines, different combination strategies have been used, such as inclusions of proteins, virus vectors and immunomodulator molecules. The best results have often been achieved when combining DNA vaccines with live vector-based vaccines. Poxviruses are effective expression vectors and are known to induce cellular and humoral immune responses in both animal models and humans. Modified vaccinia Ankara (MVA) is a highly attenuated vaccinia virus and has safely been used as a smallpox vaccine (Mayr & Danner, 1978) and in a preclinical study in immune-suppressed macaques (Stittelaar et al., 2001
). MVA is replication-deficient in human cells, and has enhanced immunogenicity because of modification of certain immune evasion genes (Blanchard et al., 1998
; Drexler et al., 1998
). A recent study in rhesus macaques has shown that repeated rMVA immunizations alone elicited lower levels of cytotoxic T lymphocyte (CTL) but higher levels of antibodies than a DNA prime/MVA boost regimen. However, both strategies led to similar control of a virulent mucosal simian-human immunodeficiency virus (SHIV) 89.6P challenge (Amara et al., 2001
, 2002
).
The ability of a multiprotein DNA/MVA vaccine, or rMVA only, to control intrarectal exposure of highly pathogenic immunodeficiency virus challenge is promising, as HIV infection is primarily sexually transmitted. Therefore, a highly desirable feature of an AIDS vaccine would be to induce mucosal immunity as a first line of defence against infection. Different routes of administration and methods of delivery of the immunogen have been shown to influence the elicited immune response. Parenteral immunizations, either by intravenous, intramuscular, subcutaneous or intradermal injections of non-replicating antigens, induce mostly systemic immune responses, whereas mucosal delivery of antigens is able to trigger both mucosal and systemic immunity. When antigens are delivered mucosally an adjuvant is generally required to generate appropriate immune responses. In rhesus monkeys mucosal immunization with an AIDS peptide vaccine formulated with an Escherichia coli toxin elicited mucosal T-cell responses and reduced virus disease after mucosal SHIV-Ku2 challenge (Belyakov et al., 2001). Besides topical application, delivery of antigen mucosally can be done simply and rapidly by high-pressure jet injectors. Intra-oral jet injection of HIV-1 DNA in solution has been shown to induce mucosal IgA in mice and activation of T cells in humans (Lundholm et al., 1999
, 2002
). Here, we explored the influence of different routes of delivery of a DNA prime and MVA boost regimen on inducing protective immunity against a homologous intravenous SHIV challenge. One group of monkeys was immunized by intramuscular needle injections combined with intra-oral and intrarectal jet injections, and another group of monkeys was immunized i.m. with only needle injections. Animals vaccinated mucosally as well as i.m. had better immune responses and control of the virus.
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METHODS |
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Immunogens.
The plasmids expressing single genes contained the HIV-1 nef (HXB3), tat, rev, RT, p37 gag (HXB2) and gp160 (BRU) genes under the control of the human cytomegalovirus (CMV) immediate early promoter, and have been described previously (Boshart et al., 1985; Hinkula et al., 1997
; Isaguliants et al., 1999
; Okuda et al., 1995
).
Recombinant MVAs separately expressing the coding sequences for HIV regulatory proteins Tat, Rev and Nef were constructed following previously described methodology (Drexler et al., 1999). Briefly, HIV-1 LAI nef (kindly provided by Marie-Paule Kieny, Transgene, Strasbourg), tat and rev (a gift from Kai Krohn, University of Tampere) were inserted into the MVA vector plasmid pUCII LZdel P7.5 to be placed under transcriptional control of the vaccinia virus early late promoter P7.5. Transfection of these plasmids into MVA-infected chicken embryo fibroblast cells (CEF) allowed isolation of the viruses MVA-HIV-1LAInef, MVA-HIV-1LAItat and MVA-HIV-1LAIrev, which were cloned by plaque purification on CEF screening for transient co-expression of the E. coli lacZ marker gene. Another recombinant MVA expressing the gagpol coding sequences of SIV mac251 32H clone pJ5 (Rud et al., 1994
) was previously described (Nilsson et al., 2001
), and served as a vaccine for the delivery of SIV Gag-Pol antigens.
Aliquots of virus preparations contained 5x108 infectious units ml1 and were stored at 70 °C. The vector vaccine preparations were tested in tissue culture infections for their capacity to synthesize HIV-1 LAI or SIV macJ5 antigens monitoring cell lysates for Nef, Tat, Rev or GagPol proteins by Western blot analysis (data not shown). Vaccinia HIV-1 BRU gp120 65.0 was constructed by Albert Osterhaus (now at Erasmus Medical Centre, Rotterdam) and Jaap Goudsmit, (now at Crucell, Leiden), the Netherlands.
Immunizations and SHIV infection.
Table 1 shows more detailed information on the immunization regimen. Four monkeys (group A) were immunized with 500 µg of each DNA (HIV-1 nef, tat, rev, gag, RT, gp160) construct intramuscularly (i.m.) at month 0 and intrarectally (i.r.) and intra-orally (i.o.) at 2 months. These immunizations were followed by 108 p.f.u. of each recombinant MVA (HIV-1 nef, tat, rev and SIV gag/pol) and 500 µg of each DNA (HIV-1 rev, env) together with 250 µg DNA (HIV-1 gag) i.m. at 4 months and the same MVA recombinants and vaccinia HIV-1 env i.r. and i.o. at 8 months. Because of difficulties in obtaining DNA expressing SIV gag/pol, we used DNA expressing HIV-1 gag and RT. No recombinant MVA expressing HIV-1 env was available, therefore we used DNA HIV-1 env for the third immunization and a vaccinia HIV-1 env for the fourth immunization. Another group of four monkeys (group B) was immunized with the same antigens i.m. at 0, 2, 4 and 8 months. Granulocyte-macrophage colony-stimulating factor (GM-CSF, 75 µg; Leukomax) was given 15 min before DNA vaccination and at the same location to all macaques at 2 months. A control group of four monkeys was given a control plasmid pkCMV i.m. at month 0 and i.r. at 2 months, and non-recombinant MVA F6(28.1) i.m. at 4 and 8 months. The i.r. and i.o. immunizations, three to five 0·2 ml injections for each dose, were delivered with a SyriJet Mark II jet injector (Mizzy). The i.o. jet injections were placed bilaterally in the cheeks in the oral cavity. The i.m. injections were given with needle injections in the hind leg.
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Preparation of peripheral blood mononuclear cells (PBMC).
PBMC were isolated from EDTA blood using FicollPaque (Pharmacia-Upjohn) gradient centrifugation and either used immediately or cryopreserved in liquid N2.
Stimulation of PBMC for enzyme-linked immune spot (ELISPOT) assay.
PBMC were either infected with recombinant poxviruses or stimulated with antigen or mitogen in tubes for 12 h at 37 °C in 7·5% CO2 prior to addition to the plates.
The following poxviruses were used for infection: MVA expressing HIV-1 (LAI) tat or nef, ALVAC HIV-1 encoding gp120 (MN); transmembrane anchor regions of gp41 (IIIB); Gag and protease (IIIB); Nef CTL domains; pol CTL epitopes (IIIB) (vCP 300), ALVAC SIV encoding gag/pol (mac142) (vCP172) at m.o.i. 5, 2 and 5, respectively. MVA wild-type and parental ALVAC were used at m.o.i. 15 to determine reactivity against the vector.
T-helper cell responses were measured using the following antigens: HIV-1 IIIB and HIV-26669 whole virus lysate (Biberfeld et al., 1988) at a concentration of 5 µg ml1 and recombinant gp120 (LAV) (Protein Sciences) at a concentration of 0·5 µg ml1. Because of cross-reactivity with SIV, HIV-2 viral lysate was used in this study.
For peptide stimulation, PBMC were pulsed directly with the peptide pools in the plate at a final concentration of 2·55 µg ml1 per peptide. The HIV-1 LAI nef (124), tat (112), env 1 (140), env 2 (4180), env 3 (81122) peptide pools consisted of 15 mers overlapping by 8 aa (aa sequences derived from pKCMVnef, pKCMVtat and pKCMVgp160 B plasmids; Kjerrstrom et al., 2001; Ljungberg et al., 2002
). The synthetic peptides above were purchased from Thermo Hybaid. The SIV gag (ARP 714.122, EVA 775.113, EVA 776.114) and SIV nef (EVA 777.125) pools consisted of 20 mers overlapping by 10 aa (kindly provided by Harvey Holmes, Programme EVA). Peptides were solubilized in DMSO at 10 mg ml1 and stored in small aliquots at 70 °C. Medium with DMSO corresponding to the highest concentration in the peptide solution was used as a mock control. Depletion of CD8+ T cells was accomplished by incubating the PBMC with magnetic beads covered with anti-CD8mAb (Dynabeads). Two cell fractions were obtained after CD8-depletion, one depleted of CD8+ T cells and the other containing CD8+ T cells+beads (enriched CD8+ T cells).
IFN- ELISPOT assay.
ELISPOT was used to determine the frequency of antigen-specific IFN--producing cells as described previously (Makitalo et al., 2002
). Responses were considered to be positive if the number of spot-forming cells (SFC) was at least twice that of the control, and >50 SFC per 106 PBMC.
Proliferation assay.
Freshly isolated PBMC (1·5x105 cells per well) were cultured in triplicate with or without antigen or phytohaemagglutinin (PHA) in complete medium in 96-well flat-bottom plates at 37 °C in 7·5% CO2 for 6 days, and thereafter pulsed with 1 µCi (37 kBq) per well of [3H]thymidine for 6 h. The antigens used were whole virus lysate of HIV-1 IIIB and HIV-26669 and recombinant HIV-1 gp160 (Protein Sciences) at final concentrations of 5 and 0·5 µg ml1, respectively. Cultures were harvested and the thymidine incorporation was measured with a 1450 MicroBeta PLUS counter (Wallac Sverige AB). Stimulation index (SI) was calculated by dividing mean [3H]thymidine incorporation in antigen-stimulated wells by mean incorporation in control wells. SI=2·0 was considered positive when proliferation against HIV-2 whole viral lysate was measured. For HIV-1 IIIB whole viral lysate and HIV-1 gp160, responses were defined as positive when SI exceeded 3·0 and 5·0, respectively (Berglund et al., 1997).
Effector cells.
For stimulation of effector cells, autologous B-lymphoblastoid cell lines (BLCL) were infected separately with each recombinant poxvirus, MVA encoding HIV-1 tat and HIV-1 nef, ALVAC (vCP 300) and SIV gag/pol (vCP172) at m.o.i. 5, 2 and 5, respectively. After infection the cells were washed and incubated overnight at 37 °C. The next day the infected BLCL were pooled, washed and fixed with 1·5% (w/v) paraformaldehyde in PBS for 15 min and thereafter treated with 0·2 M glycine in PBS for 15 min. In one experiment, effector cells were stimulated with BLCL infected with ALVAC vCP300 only. Stimulators were washed three times and added to monkey PBMC at a responder : stimulator cell ratio of 15 : 1. After 24 days recombinant human interleukin 2 (rhIL2, 40 U ml1) was added to the cells. On day 7 the cultures were restimulated by addition of fixed autologous stimulator cells as above.
When peptide-sensitized PBMC were used, PBMC (5x106) were prepulsed with an HIV-1 Tat peptide pool (EVA 770CS) for 6090 min in 100 µl complete medium, then diluted up to 2·5x106 cells ml1 in complete medium (10 µg final peptide concentration ml1) supplemented with 25 ng rhIL7 ml1 (R&D Systems). After 24 days rhIL2, 40 U ml1, was added to the cells. Fresh medium containing rhIL2, 20 U ml1, was added twice weekly to all cell cultures and CTL activity was measured after 1216 days.
51Cr release cytotoxicity assay.
Recombinant poxvirus-infected or peptide-sensitized autologous BLCL were used as target cells. For peptide-sensitized targets, BLCL were enumerated and pulsed with peptide pools as described above. As a mock target, BLCL were pulsed with medium containing 0·2 % DMSO. A standard chromium release assay was used with different effector : target cell ratios (12 : 125 : 1). Percentage-specific lysis was calculated as [(sample releasespontaneous release)/(total releasespontaneous release)]x100. In assays where MVA-infected targets were used, a 40-fold excess of unlabelled MVA (0·1 m.o.i.) infected targets (cold targets) was added to the cultures to reduce vaccinia virus-specific cytolysis present in the effectors, as previously described (Nilsson et al., 2001). Antigens used were the same as described above for stimulation of effector cells with the addition of MVA wild-type and parental ALVAC for control of vector reactivity.
Antibody assays.
A commercial microparticle enzyme immunoassay (IMx HIV-1/HIV-2 III Plus; Abbott) was used for detection of antibodies to HIV-envelope (gp41 and gp120) in serum according to the manufacturer's instructions. Serum samples were tested for IgG responses to the HIV-1 regulatory proteins Nef and Tat (kindly provided by Jonathan Karn, Medical Research Council) using ELISA as described elsewhere (Hinkula et al., 1997; Putkonen et al., 1998
).
Collection of rectal washes and detection of HIV-1 IIIB, gp120- and SIV p27-specific IgG and IgA by ELISA was performed as described previously (Nilsson et al., 2001). Native Galanthus nivalis agglutinin purified HIV-1 IIIB gp120 (Gilljam, 1993
) and recombinant SIV mac p27 (kindly provided by I. Jones through the NIBSC's centralised facility for AIDS) were used at 1 and 0·5 µg ml1, respectively, for coating. Saliva samples were collected using two different collection devices according to the manufacturers' instructions (Omnisal and OraSure). To enhance detection of virus-specific IgA, IgG was removed by use of protein A Sepharose (PharmaciaAmersham) prior to analysis. A buffer containing a protease inhibitor cocktail (Sigma) was used and all purification steps were performed on ice.
Assays for detection of virus and virus load determination.
For virus isolation, monkey PBMC were co-cultivated with PHA-stimulated human PBMC as previously described (Nilsson et al., 1995). Primers in the long terminal repeat and gag region of HIV-2 and in the env region of HIV-1 were used for the nested PCR (Walther-Jallow et al., 1999
). Levels of viral RNA in plasma samples were measured using a quantitative competitive RT-PCR assay with a lower detection limit of 40 RNA equivalents ml1 plasma (ten Haaft et al., 1998
).
Statistical analysis.
Plasma virus loads taken 2, 4 and 8 weeks post-challenge were compared between groups by MannWhitney U-test for non-parametric observations. A value of P<0·05 was considered significant.
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RESULTS |
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Group A monkeys, immunized both i.m. and mucosally, mounted earlier and higher responses to HIV-1 whole viral lysate than the animals in group B, immunized i.m. only. After only two DNA immunizations positive IFN- responses were detected in two animals (D71 and D72), and after the second MVA boost also in a third animal (D74). In group B animals, positive responses were detected in PBMC against HIV-1 lysate in one monkey (D75) following the first MVA boost and in two monkeys (D76 and D78) after the second MVA boost (Fig. 1
). No IFN-
response to HIV-2 lysate could be detected at any time point in animals in group A, and only one monkey (D76) in group B showed a weak HIV-2-specific IFN-
response (72 SFC per 106 PBMC) after the second MVA boost (data not shown). IFN-
secretion in response to rgp120 was detected in PBMC from only two monkeys (D71 and D72) (272 and 240 SFC per 106 PBMC) in group A, after the second DNA immunization and also after MVA boost (112 and 176 SFC per 106 PBMC) (data not shown).
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CTL assay.
CTL responses against MVA HIV-1 Tat, ALVAC HIV-1 gp120; Gag/Pol; Nef (vCP300) and ALVAC SIV Gag/Pol (vCP172) were measured using cryopreserved PBMC stimulated with formaldehyde-fixed autologous B-cell lines infected with corresponding poxvirus constructs.
Only one animal (D71) in group A, immunized i.m. and mucosally, showed an HIV-1 Tat-specific CTL activity after MVA boost (Fig. 2a). In animals immunized i.m. only, two monkeys had CTL activity against Tat, D76 appearing after the first DNA inoculation and D75 appearing after the MVA boost (Fig. 2b
). As the sensitivity of the CTL assay may have been reduced by the use of MVA vectors and cold targets, a pool of Tat peptides was used to assess cytotoxic activity in PBMC from four monkeys 14 days before challenge. One animal (D71) showed high specific lysis to Tat peptides at that time point and another animal (D75) showed weak reactivity (although high background), while the other two monkeys (D72 and D74) showed no specific reactivity (Fig. 2c
). This was consistent with the earlier findings in these animals of CTL reactivity against target cells infected with MVA HIV-1 Tat (Fig. 2a, b
).
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IFN- ELISPOT.
PBMC infected with poxvirus vectors ALVAC HIV-1 (vCP300), SIV (vCP172) and MVA HIV-1 Tat were also assessed for their ability to secrete IFN- in an ELISPOT assay. Only animals immunized both i.m. and mucosally showed IFN-
production in response to vCP300 (Fig. 3a, b
). Positive responses were shown in one animal (D74) after the first DNA inoculation, and in two animals (D71 and D72) after the second DNA inoculation. Following the third and fourth immunizations, three animals (D72, D73 and D74) in group A continuously produced IFN-
in response to vCP300. Depletion of CD8+ T cells demonstrated that the IFN-
response detected in the poxvirus-based ELISPOT assay was CD8+ T-cell-dependent (data not shown).
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Overlapping 1520 mer peptide pools of HIV-1 Tat, Nef and Env and SIV Gag and Nef were used to measure IFN- secretion in PBMC 2 months after the third immunization. The number of monkeys responding to the peptide pools were: group A, 3/4 to HIV-1 Tat; 4/4 to HIV-1 Nef; 4/4 to any of the HIV-1 Env pools; 1/4 to SIV Gag and 1/4 to SIV Nef; group B, 1/4 to HIV-1 Tat; 3/4 to HIV-1 Nef; 1/4 to any of the HIV-1 Env pools; 3/4 to SIV Gag; 2/4 to SIV Nef.
All four monkeys in group A responded to at least three out of seven peptide pools with a range of 57697 SFC per 106 PBMC (Fig. 4). Three monkeys in group B showed responses of a lower magnitude ranging from 52155 SFC per 106 PBMC.
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Humoral immune responses
HIV-1 Env-specific serum antibodies were measured by a commercial microparticle enzyme immunoassay prior to and 2 weeks after each immunization. Monkey D71 was the only animal that developed antibodies to HIV-1 envelope proteins, which appeared after the second DNA immunization (signal/cut off=3·30) and were further boosted after the following immunizations (signal/cut off=7·92 and 14·28, respectively).
Serum antibodies to HIV-1 Nef and Tat were investigated by ELISA at 1 and 2 months after the first MVA and 2 weeks after the second MVA immunization. Weak systemic IgG responses against the regulatory HIV-1 proteins Nef and Tat were seen in sera from all vaccinated monkeys after the first MVA. In two animals (D71 and D74), a moderate increase of antibodies to Nef was seen after the second MVA immunization (Fig. 5). The presence of specific IgG and IgA antibody responses to HIV-1 gp120 and SIV rp27, in saliva and rectal washes collected 14 days after each immunization, was determined by ELISA. No antigen-specific IgG or IgA antibodies of mucosal origin were observed in the immunized animals.
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Virus was isolated from PBMC 1 month post-challenge in 2/4 monkeys in group A and 3/4 monkeys in group B. At 2 months all vaccinated monkeys were isolation-negative, whereas 5/8 controls were still virus isolation-positive. Proviral DNA was demonstrable at 6 months post-challenge in all animals except in the only protected monkey (D71). The low replication capacity of this non-pathogenic SHIV-4 stock has been described previously (Quesada-Rolander et al., 1996).
Comparison of the immune responses at challenge with the plasma RNA levels 2 weeks post-challenge demonstrated a trend of correlation between type of immune responses and protection (Table 2; Fig. 6
). The completely protected monkey (D71) immunized i.m.+i.r.+i.o. showed a broad cellular immune response including lymphocyte proliferation, IFN-
production and CTL, and it also had antibody responses. Another monkey (D72) in the same group with the highest levels of CD8+ T-cell-dependent IFN-
responses and strong lymphocyte proliferative responses displayed no plasma viral RNA at any time point. In contrast, monkey (D77) (i.m.) with the highest virus load of all vaccines had only a lymphocyte proliferative response prior to challenge.
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DISCUSSION |
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The stronger immune responses and the more efficient virus control in animals immunized both parenterally and mucosally, in contrast to animals immunized mucosally only, may be a function of improved delivery of the plasmid and recombinant poxvirus antigens. Besides i.m. needle injection, we used a high-pressure jet injector (Syrijet) to immunize monkeys i.o. and i.r. The jet injection allows more cells to be targeted with DNA plasmids, and the mucosa provides a target tissue which contains large amounts of antigen-presenting cells and immunocompetent cells. In a previous study, mucosal jet injection of plasmid DNA in HIV-1-infected individuals triggered a Th-1-like immune response both locally and systemically (Lundholm et al., 2002). Delivery of DNA i.m. with a similar jet-injection device (Biojector) was more effective in inducing immune responses and protection against a malaria vaccine than i.m. needle injection (Rogers et al., 2001
; Wang et al., 2001
). In another study with a multiprotein DNA/MVA vaccine, the Biojector was used to deliver DNA i.m. and intradermally in rhesus macaques and induction of high levels of virus-specific T cell responses was demonstrated (Amara et al., 2001
). Although no complete protection was seen after a mucosal challenge, the elicited immune responses in those monkeys controlled the highly pathogenic SHIV-89.6P.
The monkeys in our study immunized both i.o. and i.r. received 6 mg DNA, 3 mg at each location, while the i.m. immunized monkeys received 3 mg. The higher dose may have increased the immune responses in this group of monkeys, although we believe that the route and method of administration may be of equal importance. In the study by Amara et al. (2001) two different doses of DNA were used and, while the ten times higher dose increased both the cellular and humoral immune responses, no significant effect on protection was observed.
All the immunogens used in our study appeared to be immunogenic, although the use of HIV-1 Gag and RT in the DNA immunization and SIV Gag/Pol in the MVA may explain the limited responses against these antigens.
In our study, no mucosal antibodies could be detected in either group of monkeys and mucosal CTL activity was not investigated. An intravenous challenge was used as no SHIV stock was available for mucosal challenge. Thus, future studies will address the ability of this immunization strategy to induce mucosal T-cell responses and protection against a mucosal challenge.
The importance of cellular immunity for containing virus replication in humans and monkeys has been demonstrated in several studies. Emergence of virus-specific CTL has been demonstrated to contain HIV-1 and SIV replication during primary viraemia (Koup et al., 1994; Kuroda et al., 1999
). CTL escape mutants have been detected in primary HIV-1 infection (Borrow et al., 1997
). Furthermore, monkeys temporally depleted of CD8+ T cells were unable to control virus replication both during primary and chronic SIV infection, and during primary SHIV infection (Jin et al., 1999
; Matano et al., 1998
; Schmitz et al., 1999
). Strong HIV-1-specific CD4+ T-helper responses are associated with control of viraemia in HIV-1-infected patients (Rosenberg et al., 1997
, 2000
). CD4+ T-helper cells are also necessary for the maintenance of functional virus-specific CD8+ T-cell responses (Heeney, 2002
).
The steady-state virus load is usually predictive of subsequent clinical course and outcome in both SIV-infected monkeys and HIV-1-infected humans (Mellors et al., 1996; ten Haaft et al., 1998
; Watson et al., 1997
). Others have shown a relation between the peak virus load during primary infection and the steady-state virus load, which has been demonstrated in both monkeys and humans (Lifson et al., 1997
; Lindback et al., 2000
). Because of the relatively rapid clearance of viraemia in our study, we used the plasma virus load at 2 weeks post-challenge near the peak of viraemia for analyses of correlation of protection.
The animals with the lowest virus loads in our study demonstrated broad and strong immune responses, while animals displaying the highest virus load showed more restricted and lower immune responses. These results suggests that: (i) broad immune responses including CD8+ and CD4+ T cells as well as antibodies are more efficient in controlling virus replication than cellular immune responses alone; (ii) a combination of CD8+ and CD4+ T-cell responses is more efficient than either CD8+ or CD4+ T cells alone; (iii) CD8+ T-cell responses are more efficient than CD4+ T-cell responses (CD8+ CD4+ antibodies >CD8+ CD4 >CD8 >CD4). Similar broad immune responses, consisting of both functional CD8+ and CD4+ T cells and antibodies, have been demonstrated to correlate positively with protective efficacy to HIV or SIV infection as well as disease (Hel et al., 2002; Mooij & Heeney, 2001
).
Sterilizing immunity can be obtained by administration of envelope antibodies, and it has been demonstrated that passively transferred anti-HIV-1 neutralizing monoclonal antibodies completely protected macaques against systemic as well as mucosal SHIV-89.6P challenge (Baba et al. 2000; Mascola et al., 1999
, 2000
). In a recent study, it was shown that HIV-1-specific T-cell responses induced by a DNA gag/env vaccine were unable to assist humoral immunity to prevent the establishment of initial infection, although they could reduce virus replication (Mascola et al., 2003
). Current AIDS vaccines mainly eliciting cellular immunity do not, in general, induce sterilizing immunity, but do reduce virus loads. The limitations of these vaccines have been indicated in recent studies. Barouch et al. (2002
, 2003)
showed a breakthrough of virus replication after mutations in immunodominant CTL epitopes in monkeys with a previous effective vaccine-elicited immune control of SHIV-89.6P challenge virus replication. These data suggest that an effective AIDS vaccine should induce neutralizing antibodies as well as functional CD4+ T cells and CD8+ T cells. It is likely that a vaccine able to elicit broader immune responses against many virus proteins will exert more effective control of virus replication than a vaccine inducing a narrow immune response.
The protected monkey (D71) together with two monkeys immunized i.m. (D75 and D76) had CTL against Tat, and they appeared to have an earlier control of virus replication than the other monkeys in that group. CTL activity against proteins expressed early in HIV-1 replication cycle (Tat, Rev and Nef) may be more beneficial than CTL against late structural proteins in controlling infection (Calarota et al., 2001; Gruters et al., 2002
). In studies in humans and monkeys, virus specific CTL against Rev and/or Tat has been shown to correlate with control of virus replication (Cafaro et al., 2001
; Osterhaus et al., 1999
; van Baalen et al., 1997
). However, data published recently suggest that immunization with only Rev and Tat vaccines is not sufficient to achieve solid protection (Allen et al., 2002
; Verrier et al., 2002
).
A peptide-based DNA/MVA HIV vaccine designed to elicit mainly CD8+ T-cell responses has already entered clinical trials in Oxford and Nairobi. This vaccine has been shown to induce T-cell responses in rhesus macaques (Wee et al., 2002), and preliminary studies show induction of T-cell responses in a majority of human volunteers by either vaccine component alone (Hanke et al., 2002
). In contrast to our vaccine, the vaccine used by Hanke and McMichael consists of a consensus HIV clade A p24/p17 and a string of CTL epitopes (Hanke & McMichael, 2000
).
To increase the efficacy of DNA-based vaccines, different cytokine adjuvants have been tried. In mouse models, co-immunization of DNA vaccines with GM-CSF plasmids has been reported to enhance immunogenicity and protective efficacy of malaria DNA vaccines (Weiss et al., 1998). Both groups of monkeys in our study received GM-CSF protein just before the second DNA immunization. Thus, no conclusions about the effect of GM-CSF can be drawn from this experiment. In one recent paper, the use of plasmids expressing IL12/GM-CSF in addition to an SIV DNA prime/virus-like particle protein-boost strategy enhanced protection against mucosal SIVsmE660 challenge in monkeys (O'Neill et al., 2002
). However, in another study of monkeys genetically immunized with an SIV library vaccine and challenged intravenously with SIV mac251, the use of plasmids encoding IL12 and GM-CSF resulted in lower survival rates (Sykes et al., 2002
). These results show that more work is needed to determine the effect of GM-CSF as a cytokine adjuvant.
Our results demonstrate that cellular immune responses were elicited by immunization of macaques with a multicomponent DNA/MVA vaccine. The administration of the vaccine by mucosal injection both i.o. and i.r. in combination with the i.m. route was shown to be more effective in inducing cellular immune responses and a better control of challenge virus replication than i.m. injection alone. In addition, the breadth of the elicited immune response correlated with control of infection in the vaccinated monkeys. Based on this concept of a DNA prime-MVA boost strategy, a multiprotein DNA/MVA HIV vaccine will enter clinical phase I trials in the near future.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 2 December 2003;
accepted 30 March 2004.