Division of Retrovirology, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Herts EN6 3QG, UK1
Author for correspondence: Neil Almond.Fax +44 1707 649865. e-mail nalmond{at}nibsc.ac.uk
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Abstract |
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Introduction |
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It is possible that the failure of vaccines based on monotypic envelope is due to the rapid evolution and selection of escape variants in the immunized host following challenge. The genetic evolution of SIVmac in macaques is very similar to that of HIV-1. Viruses expressing distinct env sequences appear readily, even when the initial infection is with virus prepared from infectious molecular clones (Burns & Desrosiers, 1991 ; Johnson et al., 1991
). There is evidence that the rate of mutation and in vivo selection is accelerated in macaques that have been immunized with Env-based vaccines prior to challenge with uncloned virus stocks (Almond et al., 1993
; Polacino et al., 1999
). Thus, the simian model is well suited to determine whether monotypic envelope vaccines drive this process of mutation and selection in macaques following challenge with cloned virus stocks.
To address this hypothesis, six macaques were immunized with recombinant SIV Env (gp120) with or without priming with recombinant vaccinia virus expressing SIV Env. After challenge with SIV derived from an infectious molecular clone, sequences of env genes from recovered viruses were analysed for evidence of immune selection.
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Methods |
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Recombinant vaccines.
The construction of vaccinia virus expressing recombinant (r) gp160 derived from the SIVmac molecular clone BK28 has been described previously (Horth et al., 1991 ). Recombinant gp120, derived from the molecular clone BK28, was produced in Drosophila cells and purified by using lentil lectin. For immunization, it was mixed with the Syntex emulsion SAF-1 containing threonyl muramyl dipeptide as described previously (Mills et al., 1992
).
Serological responses.
Antibodies that bound SIV Env were determined by ELISA with recombinant SIV gp140 (Repligen Corporation) as antigen, as described previously (Stott et al., 1990 ).
The capacity of the serum to neutralize a stock of SIVmac J5 in vitro was determined as described previously (Kent et al., 1994 ). It is presented as the reciprocal dilution of heat-inactivated serum capable of inhibiting mean SIV p27 antigen production by 75% following infection of C8166 cells.
Proliferative T-cell responses.
Proliferation of PBMC following stimulation with recombinant SIV gp120 was determined as described previously (Jones et al., 1992 ).
Virus recovery from PBMC and the assessment of virus load.
Virus re-isolation from the blood of macaques was performed by co-culture of 5x106 PBMC with 106 C8166 cells. The presence of CPE was confirmed by an antigen-capture assay specific for SIV Gag protein (Rose et al., 1995 ). To determine the virus load, tenfold dilutions of PBMC (106 to 102) from each macaque were co-cultivated in duplicate, in 24-well plates, with 105 C8166 cells.
Diagnostic PCR and quantification of the SIV provirus burden.
The extraction of DNA from the blood of macaques and the diagnostic amplification of the SIV gag gene have been described previously (Rose et al., 1995 ). The SIV provirus load was determined by combining end-point dilution of DNA with PCR (Slade et al., 1995
). The number of SIV genomes per 106 cells was calculated on the basis that 6·6 µg total DNA is equivalent to 106 cells.
Nucleotide sequencing of SIV env.
The amplification of the entire gp120 region of SIV env by PCR and the direct determination of the nucleotide sequence of the PCR product has been described previously (Almond et al., 1992 ).
Experimental design.
Group A: three cynomolgus macaques (K79, K89 and K90) received four subcutaneous immunizations of 85 µg purified rgp120 protein with adjuvant on weeks 0, 4, 8 and 104. Group B: on weeks 0 and 4, three cynomolgus macaques (K83, K84 and K94) were inoculated intradermally with 2x108 p.f.u. of the recombinant vaccinia virus expressing the SIV Env (gp160). At weeks 8 and 104, these macaques were boosted by subcutaneous injection of 85 µg purified rgp120 formulated with adjuvant.
Two weeks after the final boost (week 106), all six immunized macaques, as well as four naive controls (group C: M77-M80), were challenged by intravenous inoculation with 10 monkey infectious doses (MID50) of the 3/92 stock of the SIV molecular clone J5 prepared on simian cells (also known as J5M; Rud et al., 1994 ). The animals were sacrificed between 8 and 10 weeks after challenge.
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Results |
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T-cell proliferative responses were assessed in selected macaques from each group throughout the course of immunization. Fourteen days after the third immunization, a significant specific proliferative response [stimulation index (SI) 2·0] to SIV Env was detectable in all three macaques tested (K89, SI=5·2; K90, SI=2·3; K94, SI=9·4). On the day of challenge, proliferative responses to SIV Env were assessed for all six immunized macaques. A specific response was detected only in macaque K84 (SI=2·4).
Detection of virus after challenge
Nine of the ten macaques became infected after challenge with SIVmac J5M, as determined by co-cultivation of PBMC (Table 2). Virus was recovered from every sample taken from control macaques at 2, 4 and 8 weeks post-challenge. The recovery of virus from immunized macaques was more sporadic. Virus was not recovered from K83 and K90 at 2 weeks post-challenge or from K89, K90 and K94 at 8 weeks post-challenge. Virus was never reisolated from the PBMC of macaque K84.
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Virus burden by co-culture and DNA PCR
The virus load in PBMC was determined at 2 weeks post-challenge (Table 1). In all naive controls (M77-M80) and two of the immunized macaques (K79 and K89), the virus load was greater than 1000 infected cells per 106 cells cultured. For macaque K94, the virus load was 100-fold lower. No virus was co-cultured from 2x106 PBMC of macaques K84, K89 or K90, although virus was recovered from 5x106 PBMC of K89 and K90.
The SIV DNA burden in the blood at 2 weeks p.i. was determined by a combination of end-point dilution and PCR (Table 1). The provirus burden in the control macaques (M77-M80) ranged between 9900 and 210000 copies per 106 cells. The provirus burden in the blood of immunized macaque K79 fell within this range. In all other immunized macaques, the provirus burdens were lower (range <152400 copies per 106 cells). For macaques in group B, which had been primed with recombinant vaccinia virus, the provirus burdens were significantly lower than in controls (P<0·05 by Student's t-test).
Relationship between virus burden and antibody response
There was no significant correlation between ELISA antibody titres at challenge and reduction in virus load at challenge (Table 1) (Spearman's rank correlation coefficient; r=-0·30; P=0·57). Although a group A macaque, K90, with the highest neutralizing antibody titre, had the lowest virus load, this was certainly not the case in group B, where macaque K84 had no detectable neutralizing activity but was apparently protected from detectable infection.
Sequence of SIV env
The gp120 region of SIV env was amplified by nested PCR from DNA extracted from the blood of immunized macaques K79, K89, K90, K83 and K94 and control macaques M77, M78 and M80 at 2 weeks post-challenge. The nucleotide sequence of each PCR product was determined from twelve amino acids upstream of the V1 region to fourteen amino acids from the gp120:TM protein cleavage site. The predicted amino acid sequences obtained from nucleotide sequencing are shown in Fig. 2. Changes in the nucleotide sequence that did not alter the predicted amino acid sequence were detected in two macaques, K90 and K83 (Fig. 2
). Only two macaques, immunized macaque K90 and control macaque M80, exhibited any differences. In control M80, the amino acid difference was at amino acid position 424, in the V4 region (equivalent to amino acid 427 of BK28 Env), where a nucleotide sequence ambiguity changed the histidine residue to a mixture of histidine and tyrosine residues. In immunized macaque K90, the change was at amino acid 323, in the V3 loop equivalent (amino acid 326 of BK28 Env), changing the predicted amino acid from isoleucine to serine. This identical change was observed in the predicted sequence of two independent PCR products amplified from the same DNA template. However, sequence analysis across this V3 equivalent region of PCR product amplified from the blood of K90 at 4 weeks p.i. found that the only detectable sequence was identical to that of the infecting virus, J5.
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Discussion |
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This study failed to correlate antibodies with protective immunity in the macaque. Titres of ELISA antibody to Env were higher in macaques receiving the combined immunization protocol of vaccinia virus priming and boosting with recombinant Env in adjuvant than in animals receiving purified Env protein in adjuvant alone. Previous studies have produced contradictory evidence for a role for antibody in vaccine protection against SIV (Almond et al., 1997 ; Gardner et al., 1995
; Haigwood et al., 1996
; Kent et al., 1994
; Putkonen et al., 1991
). It is widely believed that neutralizing antibodies are a useful immune correlate of protection by lentivirus vaccines (Clements et al., 1995
; Wyand et al., 1996
). In this study, however, neutralizing antibody titres on the day of challenge did not correlate with the ability to control virus load after challenge. Indeed, for macaque K84, which did not become infected after challenge, no neutralizing activity was detected in the serum at the time of virus challenge. Others have suggested that cellular immune responses are more beneficial in conferring protection and preventing progression to disease (Clerici & Shearer, 1994
; Gallimore et al., 1995
), although the evidence is inconclusive (Hulskotte et al., 1995
). Only limited analysis of cellular immunity was performed in this study, but differences were not detected between T-cell proliferative responses of the two groups of immunized macaques. However, cytotoxic T-cell responses were more likely to have developed in group B than in group A. It is intriguing that K84, which did not become infected, was the only macaque that had a significant proliferative T-cell response on the day of challenge. Although inconclusive, the contrast between the lack of neutralizing antibody and the stronger proliferative responses would reinforce the view that T-cell rather than B-cell responses are in general more beneficial.
In spite of intensive research by a number of groups, recombinant envelope vaccines have seldom conferred protection against detectable infection with SIVmac (Ahmad et al., 1994 ; Daniel et al., 1994
; Giavedoni et al., 1993
; Israel et al., 1994
). In this study, we addressed whether immune pressure resulted in the rapid selection, within 2 weeks, of variants capable of side-stepping vaccine protection. The analysis of SIV env sequences amplified from the blood of immunized and control macaques failed to detect any group-specific sequence differences, suggesting that immune escape does not account for the vaccine failure. Indeed, it was surprising that the predominant env sequence was so stable in all groups of macaques. It is possible that the results obtained by direct sequence analysis of the PCR products did not reflect the true situation in the blood, due to selective sampling from macaques with a low provirus burden. Similar problems have skewed the results of previous studies with a similar design (Slade et al., 1995
). However, this is unlikely to be the case in this study, since quantification prior to sequencing ensured that every PCR used for sequence analysis contained at least 10 copies of SIV template. Furthermore, the sequence changes detected were obtained reproducibly from independent PCR products derived from the same DNA template. Alternatively, this result may have arisen because the sequence of the challenge virus SIVmac J5M represented an immune escape variant compared with the immunogens, based on the clone SIVmac BK28. This suggestion is also unlikely, since both neutralizing antibody and proliferative T-cell responses were assessed by using virus or antigens derived from the challenge virus. Significant immunological cross-reaction was observed between the vaccine and the challenge virus. Furthermore, by definition, a vaccine escape variant would not be expected to be susceptible to the effects of immunization, whereas a significant decrease in virus load was observed in macaques from group B.
The results of this study provide a valuable comparison for recent reports of participants in phase I/II clinical vaccine trials who have become infected during or following immunization with HIV-1 Env (Berman et al., 1997 ; Connor et al., 1998
). So far, the infected vaccine volunteers studied in these reports have been recipients of subunit HIV-1 Env vaccines only and frequently had significant neutralizing antibody titres in serum before they became infected. These observations, that immunization with Env subunit vaccines has not protected recipients from infection, are very similar to those obtained from the macaques in group A that received recombinant SIV gp120 only. Nevertheless, the results from the macaques in group B confirm the observations of Hu et al. (1992)
, that a vaccine combining a poxvirus-based vaccine to prime and a recombinant HIV-1 gp120 protein boost can be more successful in the generation of effective protection. Nevertheless, the sequence analysis in this simian study suggests that we should be cautious in the interpretation of similar sequencing studies in humans. In the report from Connor et al. (1998)
, the authors concluded that the failure to detect any skewing of env sequence recovered from the infected vaccinees indicated that there was no vaccine effect. Our simian study clearly demonstrates that, even in cases where a vaccine effect (i.e. reduction in virus load) is observed, this need not necessarily result in the more rapid appearance of genetic variants of the virus.
This study indicates that the rapid appearance of immune escape variants does not account for the failure of recombinant Env vaccines to confer complete protection. The sequence stability of the env gene sequence in the blood for at least 2 weeks after challenge will renew hopes that improvements in the immunization regime of recombinant envelope vaccines may enhance their efficacy. It is possible that evaluating vaccine efficacy by intravenous challenge with SIV is too severe. This may account for the greater success reported by Lehner et al. (1996) , who reported that immunization with recombinant Gag and Env proteins targetted to specific lymph nodes draining the gut elicited protection against rectal challenge with SIVmac.
This study did not address whether the partial protection achieved in macaques in group B would improve the clinical prognosis following infection. Reports indicate that individuals who control the initial infection most effectively and have the lowest `set-point' viraemia progress to disease most slowly (Haigwood et al., 1996 ; Hirsch et al., 1996
; Mellors et al., 1996
; Watson et al., 1997
). However, a clear relationship between the circulating virus load 2 weeks post-challenge and the `set-point' viraemia at 612 weeks post-challenge has yet to be identified.
This study did not identify a reason for the failure of recombinant envelope vaccines to confer more than partial protection. Nevertheless, the results provide encouragement for the continued development and evaluation of monotypic recombinant vaccines against immunodeficiency viruses. If the failure of the vaccines to confer potent protection cannot be ascribed to the rapid mutation and selection of immune escape variants, then further work to evaluate alternative immunization regimes may prove fruitful in the quest for a safe, effective AIDS vaccine.
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Acknowledgments |
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Footnotes |
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c Present address: Wells Medical Ltd, Royal Tunbridge Wells, Kent, UK.
d Present address: National University of Ireland, Maynooth, Co. Kildare, Ireland.
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References |
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Received 10 March 1999;
accepted 30 April 1999.
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