Recombinant SIV gpl30, produced in Chinese hamster ovary cell expression systems, was obtained from the Chiron. Corporation. The sequence expressed was ...
Journal of General Virology (1993), 74, 865-871. Printedin Great Britain
865
Sequence variation in the e n v gene of simian immunodeficiency virus recovered from immunized macaques is predominantly in the V I region N . Almond, ~* A. Jenkins, 1 A. B. H e a t h 2 and P. Kitchin ~ 1A I D S Collaborating Centre and 2Informatics Laboratory, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, U.K.
Three cynomolgus macaques were immunized with recombinant envelope protein preparations derived from simian immunodeficiency virus (SIV). Although humoral and cellular responses were elicited by the immunization regime, all macaques became infected upon challenge with 10 MIDs0 of the 11/88 virus challenge stock of SIVmac251-32H. The polymerase chain reaction was used to amplify proviral SIV gpl20 sequences present in the blood of both immunized and control macaques at 2 months post-infection. A comparison of the predominant sequences found in the region from V2 to V5 of gpl20 failed to differentiate provirus recovered from either immunized or control animals. A detailed investigation of sequences obtained from the hypervariable V1 region identified a mixture of sequences in both immunized and control macaques. Some sequences were identical to those previously detected in the virus
challenge stock, whereas others had not been detected previously. Phenogram analysis of the new V1 sequences found in immunized animals revealed that they were quite distinct from those from the virus challenge stock and that they included alterations to potential N-linked glycosylation sites. In contrast, new sequence variants recovered from the control animals were closely related to sequences from the virus challenge stock. The difference in diversity of new V1 sequences recovered from immunized and control macaques was highly significant ( P < 0.001). Thus, the presence of preexisting immune responses to SIV envelope protein is associated with greater genetic change in the V1 region of gpl20. These data are discussed in relation to the epitopes of SIV gpl20 that may confer protection from in vivo challenge.
Introduction
potential vaccine preparations. However, to date there has been only one report describing the successful use of a recombinant vaccine, which contained envelope protein of a single genotype, to protect macaques against experimental infection with SIV (Hu et al., 1992). One possibility, which may account for the failure of many experimental attempts to use recombinant immunogens to confer protection, may be the variability of the retroviral genome. It has been well documented that SIV, like H1V, exists as a number of related, but genetically distinct sequences (collectively called quasispecies) which are evolving continuously and changing both in vitro and in vivo (Burns & Desrosiers, 1991; Johnson et al., 1991 ; Overbaugh et al., 1991 ; Almond et al., 1992a, b). The failure of recombinant immunogens of a single genotype to protect may be a result of the virus evading the host's anti-SIV immune responses by the emergence of new viral sequence variants. Thus, the characterization of viruses recovered from previously immunized animals may result in a better understanding of the factors that account for the failure of the specific vaccine preparations used to date. As a result, the
A number of experimental systems have been developed to model the infection of humans with human immunodeficiency virus (HIV). One application of these models is in the development of effective vaccine strategies against HIV. From studies of the infection of chimpanzees with HIV-1, it has been found that immune protection can be mediated via the viral envelope protein alone (Berman et al., 1990; Emini et al., 1992). However, extensive vaccine development using this model is limited by the restricted number of animals available. A number of groups have chosen other simian models, such as the infection of macaques with isolates of simian immunodeficiency virus (SIV) (reviewed by Desrosiers, 1988; Gardner & Stott, 1990). Using these models, a number of different immunogens have been tested as The nucleotide sequence data reported in this paper have been submitted to the GenBanknucleotidesequencedatabase and havebeen assigned the accession numbers L05111 to L05126, and L06344 to L06380. 0001-1406 © 1993 SGM
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N. Almond and others
requirements for effective vaccines may be defined more clearly. In this study, three cynomolgus macaques were immunized with a recombinant SIV envelope preparation derived from the molecular clone SIVmac2511A11 (Marthas et al., 1989). This protein, produced in Chinese hamster ovary cells, contains only the extracellular portion of the envelope protein and has an M r of 130K. The immunization protocol induced high levels of anti-envelope antibodies in the host, including high-titre neutralizing antibody against the virus stock used to challenge the animals (E. J. Stott et al., personal communication). However, upon challenge with the 11/88 virus challenge stock of SIVmac251-32H all the immunized animals became infected. In this report, the sequences of the SIV env gene recovered from the blood of the immunized animals at 2 months post-infection are described. These sequences are compared with those recovered from the blood of the control animals, as well as those present in the original challenge stock (Almond et at., 1992a). This is the first report of a vaccine trial where the sequences of the recombinant immunogen, the virus challenge stock and the 'breakthrough' virus have all been characterized. The results indicate that the greatest genetic change observed in immunized animals in vivo was restricted to the V1 region of the SIV env gene.
Methods Vaccine studies. Recombinant SIV gpl30, produced in Chinese hamster ovary cell expression systems, was obtained from the Chiron Corporation. The sequence expressed was derived from the molecular clone SIVmac215-1All, which was isolated from the virus pool SIVmac251 (Marthas et al., 1989). The immunization schedule and the characterization of the immune responses induced will be described in detail elsewhere (E. J. Stott et al., unpublished). Briefly, three cynomolgus macaques 0146, J174 and J 175) were immunized by four subcutaneous injections of 50 gg protein mixed with adjuvant SAF-1 (Stott et al., 1990) given on weeks 0, 4, 8 and 16. Two weeks after the final immunization, the animals were bled for the characterization of anti-SIV responses. The three immunized animals and three controls (J149, J150 and J151) were inoculated with 10 MID~0 of the 11/88 virus challenge stock of SIVmac. This virus challenge stock was derived from a macaque called 32H that had been inoculated with the virus pool SIVmac251 (Kitchin et al., 1990a; Cranage et al., 1990; Almond et al., 1992a). All six macaques became infected as judged by virus re-isolation and diagnostic PCR (Kitchin et al., 1990b) performed at 2 weeks post-infection.
env gene sequence analysis. At 8 weeks post-infection blood from all macaques was taken and added to 0.15 % (w/v) sodium citrate and then DNA was isolated as described previously (Kitchin et al., 1990b). Following digestion with the restriction endonuclease SalI, the gpl20 region of the SIV env gene was amplified specifically, using two rounds of the PCR and nested oligonucleotide primer pairs, as described previously (Almond et al., 1992 b; the primers for the first round were SE6449N and SE8303C and for the second round were SE6574NSE and SE8127CKE). The number of cycles in each round of amplification
was adjusted depending on whether the product was used directly for sequence analysis (40 cycles first round, 40 cycles second round), or else cloned into M13 mpl8 (10 cycles first round, 30 cycles second round) prior to sequencing (Almond et al., 1992b). Panels of recombinant clones o f gp 120 (Bg, 12 clones; B1, 12 clones; Bi, 11 clones; and Bm, 12 clones) were prepared from the product of the second round of amplification as described previously (Almond et al., 1992 a). Sequence analysis of the PCR products of the gpl20 region of the env gene were performed as described previously (Almond et al., 1992a, b). Briefly, sequencing of M13 clones was performed using Sequenase (United States Biochemical Corporation), following the manufacturer's protocol except that termination reactions were performed at 50 °C. Direct sequence analysis of PCR products using Sequenase was performed following gel filtration of the product through Sepharose CL-4B (Pharmacia) to remove PCR primers. The PCR product was boiled for 5 min in the presence of the sequencing primer and then rapidly frozen in a dry-ice/ethanol bath. Labelling reactions were performed at room temperature and termination reactions were performed at 50 °C. The deduction of nucleotide sequences from autoradiographs was performed, in some cases, using the BaseScanner (Amersham). The relationships between nucleotide sequences were analysed by constructing phenograms, or trees, using a distance matrix approach (Nei, 1987) as described previously (Almond et al., 1992a). We have found that this approach of combining the sequence analysis of clones derived from PCR products and tree analysis produces highly reproducible results as to the sequence diversity between clones obtained from independent amplifications of the same DNA template (Almond et al., 1992 c).
Results Sequence analysis of gpl2O from V2 to V5
The predominant sequence of SIV gpl20 3' of the V1 region was obtained by direct sequence analysis of PCR products, amplified from each animal (equivalent to nucleotides 7070 to 8130 of the clone SIVmac251-32H; GenBank accession number D01065). The amino acid sequence predicted from the nucleotide sequence is shown in Fig. 1. Since the sequence of gp 120 between the V2 and V5 regions was resolved, this suggests that there was a single predominant proviral sequence for this region present in the blood of each animal. Only three of the six sequences (derived from immunized macaques J146 and J174 and one control macaque, J151) showed any variation from the predominant sequence derived from the 11/88 virus challenge stock labelled 11-88pep in Fig. 1. In two of these cases (immunized macaque J146 and control macaque J151), the sequence of SIV amplified was identical to a minor sequence variant present in the original virus challenge stock. This variant is exemplified by clone W5 as described in Almond et al. (1992a). In the other immunized macaque (J174), a new envelope sequence variant predominated. In this animal, three amino acid changes were observed in the sequence around the region equivalent to the HIV-1 V3 loop (Fig. 1 I to V at amino acid 340, and K to N at amino acid 345 and K to N at amino acid 354). However, a comparison of all the amino acid sequences failed to identify any
SIV env from immunized macaques
155 J 1 4 6 ---~qn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
254 i. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
J174 .................................................................................................... J175 .................................................................................................... J149 .................................................................................................... J150 .................................................................................................... J151 - - - a q n . . . . . . . . . . . s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i . . .
Immune
Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
ll-88pep ................................................................................................... Chiron-env ---v .......................... x......... a.......... g. . . . . . . . . . . . . . . . . . . . . . . . . Consensus
SCITHDNCTGLEOEOMIGCKFNMTG
LKRDKTKEYNETWYSTDLVCEOGNS
867
TDNESRCYMNHCNTSVIOESCDKHY
a...................... WDTIRFRYCAPPGYALLRCNDTNYS
V2 255
354
J146 .................................................................................................... J174 ..................................................................................... J175 ....................................................................................................
v . . . . ~. . . . . . . .
~ Immune
J149 .................................................................................................... J150 .................................................................................................... J151 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11-88pep . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chiron-env ........................................................... i ................. Consensus ~PKcswrcvsscr~T0rs~
Control q. . . . . . . . . . . . . . . . . . .
r--
rGn~GT~EteTYI'~aGRO~rIX SU,~Yr~ZT~C~PG.KT"VL~WrX MSGLVFHSOPINDRPKOAWCWFGGK V3
355 454 J146 .................................................................................................... J174 .................................................................................................... Immune J175 ....................................................................................................
J149 ....................................................................................................
J150 .................................................................................................... J151 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Control
11-88pep . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chiron-env ................................................................... a................................ C o n s e n s u s WKDAI~EVXQTXVKnPRYrGT~STDKINLTAVGGGDPEVrV~Tr~Cr~GEVLYC~NWVL~WVEDr~r~KWEO HKRNyVPCHIRQIINTWHKVGKNVy V4 455 519 J146 ................................................................. J174 ................................................................. Immune J175 .................................................................
J149 ................................................................. J150 ....................................................
151 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
ll-88pep ................................................................. Chiron-env .......... i ..................................................
~3~25~33333
Control
n-r-
ConsensusLPPRZGDLrCNSrVTSL~AN~DW~O GNOrNXTMSAEVAELYRLE~DYXLVZXTPm~PVDVKR V5 Fig. 1. T h e a m i n o a c i d s e q u e n c e o f g p 1 2 0 f r o m the V 1 V 2 j u n c t i o n to t h e g p l 2 0 - g p 4 1 j u n c t i o n . T h i s w a s p r e d i c t e d f r o m t h e direct s e q u e n c e a n a l y s i s o f P C R p r o d u c t s a m p l i f i e d f r o m t h r e e i n f e c t e d m a c a q u e s 0 1 4 6 , J 1 7 4 a n d J175) i m m u n i z e d w i t h a r e c o m b i n a n t SIV e n v e l o p e p r o t e i n ( g p l 3 0 ) p r e p a r e d b y C h i r o n C o r p o r a t i o n p r i o r to c h a l l e n g e w i t h 1 1 / 8 8 v i r u s c h a l l e n g e s t o c k . I n a d d i t i o n , the s e q u e n c e s p r e d i c t e d f r o m t h e P C R p r o d u c t o f t h r e e n a i v e i n f e c t e d c o n t r o l m a c a q u e s (J 149, J 150 a n d J 151), as well as the p r e d o m i n a n t s e q u e n c e o f e n v e l o p e p r e s e n t in 1 1 / 8 8 c h a l l e n g e s t o c k ( 1 1 - 8 8 p e p ) a n d t h e s e q u e n c e o f the r e c o m b i n a n t e n v e l o p e p r o t e i n p r e p a r a t i o n ( C h i r o n - e n v ) a r e s h o w n . T h e c o n s e n s u s s e q u e n c e is g i v e n in single-letter c o d e a s the b o t t o m line. O t h e r s e q u e n c e s a r e s h o w n a b o v e . A d a s h i n d i c a t e s t h a t the a m i n o a c i d is i d e n t i c a l to the c o n s e n s u s s e q u e n c e ; v a r i a t i o n in s e q u e n c e is g i v e n in single-letter c o d e . T h e r e g i o n s o f the e n v e l o p e p r o t e i n e q u i v a l e n t to the v a r i a b l e r e g i o n s o f H I V - 1 a r e i n d i c a t e d ( A l m o n d et al., 1992a).
consistent variations between V2 and V5 specific to either the immunized or the control groups of infected macaques.
Sequence analysis of the V1 region of gpl20 The sequence of the V1 region of gpl20 (equivalent to nucleotides 6941 to 7070 of the clone SIVmac251-32H) was determined from panels of recombinant M 13 clones prepared from specific PCR products. These panels had been constructed for two of the immunized macaques (J146 panel prefixed Bg, 12 clones; J175 panel prefixed B1, 12 clones) and for two of the control macaques (J149
panel prefixed Bi, 11 clones; J150 panel prefixed Bm, 12 clones). This sequence analysis also included the sequence across the V2 region (equivalent to nucleotides 7070 to 7300 of the clone SIVmac251-32H), which had been determined previously by direct sequence analysis (see above and Fig. 1). The consensus sequence of the V2 region deduced from each panel was identical to that obtained from the corresponding template by direct sequence analysis. The nucleotide sequence analysis across the V1 region revealed a mixture of sequences present in both immunized and control animals. Some of the clones were identical in sequence to clones isolated from the virus
868
N. A l m o n d and others
1~
I
BI6 BIB i--819
/, ] I
,
B~5
/
I-'BI12
1 J175 immune
BIIO
t--BI7 ~ B I 4 L - - B I 1 Vlv21 al 1
B~ ~Bgl W3 I [-ag9
L
Immunogen ._J J146 immune "-] 11/88 challenge stock
--1
~--t B~7 //
/Bg12
J146 immune
[--
Bgl 0.._1 ,Bi4 J149 control r - Brn7 J150 control ~--Bm4 Bi6 Bil 1 .._._._~ Bi3 Bil Bi8 Bi7 Bi5 Bi12
Bi10
--Wl
J149 control
11/88 challenge stock
Bin9 "-'1 Bm5 / Bin3 / J150 controls Bin2 / Bin12 I Bml~/ BmlO I Bin1
m
BI3 /
BI2 / J175 immune BI11 ._] Bg6 - -
B~
BBBgg / 92
I
25
I
20
I
I
15 10 Difference between sequences (%)
I
5
J146 immune
-- Bgl 1 I
0
Fig. 2. Trees representing nucleotide sequence variation across the V1 region between clones of SIV gpl20 recovered from immunized macaques J146 (clones prefixed Bg) and J 175 (clones prefixed B1), control macaques J 149 (clones prefixed Bi) and J 150 (clones prefixed Bin) and sequences present in the 11/88 virus challenge stock (prefixed W) and the immunogen.
challenge stock, other clones had new V1 sequences not detected previously ( < 6% frequency in the virus challenge stock). Only two sequences detected in the challenge stock were re-isolated from any of the macaques in vivo. These are exemplified by clones W1 and W3 in the challenge stock (Almond et al., 1992a). The proportion of new V1 sequences differed for each macaque. For immunized macaque J 146 the sequences of 10 out of 12 clones were identical to the challenge stock sequences W1 or W3. However, for the second immunized macaque, J175, only three out of 12 clones had sequences identical to those in the challenge stock (W1). For control macaque J150, 10 out of 12 clones were identical to the challenge stock sequence W1. In contrast, for J149 none of the 10 clones had sequences which had been detected in the challenge stock.
To dissect the complex relationships between the V1 sequences recovered from immunized and control macaques, trees or phenograms were constructed. In Fig. 2 the sequences of the V1 region recovered from each animal were compared with each other, with the sequence of the immunogen and with the challenge stock sequences W1 or W3. This figure shows that none of the V1 sequences recovered in vivo resembled the sequence of the immunogen ( > 15 % sequence diversity). In addition, it confirms that a number of clones recovered from both immunized and control macaques were identical to challenge stock sequences W1 or W3. However, this tree distinguishes the new V1 sequences recovered from immunized animals and those recovered from control macaques. All of the new V1 sequences recovered from control macaques (Bm4 and Bm7, and Bil to 12) were
S I V env f r o m immunized macaques 113
156 - -
--R ..........
---
-
---~ . . . . . . . . . . . . . .
-
.............. .............. .............. ---- .............. - ---- ............. .
.
.
.
.
.
.
.
.
.
---- ..............
T.. - -
A
Immunogen
KAMSE--N-I-~---
AS. . . . . . . . .
P SE
A. . . .
A.-s . . . . . . .~ - - -
A ..... a. -s-v A ..... a. -s-vA .... ..-A. s . . . . . Zr . . . . . . TT-P-~P . . . . TT-P--XPT..- . . . . TT p -~p . . . . T T . -
.
.
.
.
zs . . . . . .
CNK~ETDKWGLTKSSTTT-PT
.
.
.
.
.
p-ss APT
-- -
11% 42 % 50% 100% 50 % 25 %
In In In In In In
challenge stock cf W3 immunized J146 challenge stock cf W l control J150 immunized J 146 immunized J175
-G~ --~ ~ S-N-s-N~_ s-~-~_
18 % In control J149 72 % In control J149 9 % In control J149 16%InimmunizedJ175 50 % In immunized J175 8 % In immunized J175
-s-N-~-
8 % In immunized J146 Consensus
VA-KIDMVNETSSC
F i g . 3. T h e a m i n o a c i d d i v e r s i t y o f t h e V1 r e g i o n o f t h e e n v e l o p e protein in immunized and naive infected macaques. This was predicted from the nucleotide sequence of clones prepared from PCR products of the SIV env
gene amplified from DNA
infection from
immunized
macaques
recovered 2 months
(J146 and
J174) and
post-
control
m a c a q u e s ( J 1 4 9 a n d J 1 5 0 ) . L a y o u t a n d s y m b o l s a r e a s i n F i g . 1. T h e proportion of clones with each particular sequence recovered from each a n i m a l is i n d i c a t e d a t t h e r i g h t - h a n d e n d o f e a c h line. A d o t i n d i c a t e s t h a t n o a m i n o a c i d is p r e d i c t e d a t t h e r e s i d u e . P o t e n t i a l sites o f N l i n k e d g l y c o s y l a t i o n are u n d e r l i n e d . S e q u e n c e s a b o v e t h e s o l i d l i n e h a v e been o b s e r v e d p r e v i o u s l y , those b e l o w are new V I sequences not detected previously.
less than 5 % divergent, at the nucleotide level, from sequences present in the challenge stock. In contrast, new V1 sequences recovered from immunized macaques (Bg8 and Bll, B14 to 10 and Bl12) were greater than 10% divergent from any challenge stock sequences. The similarities (percentages of matching nucleotide bases) of new V1 sequences to their closest challenge stock sequence were calculated, for both the immunized group and the control group. The resulting similarities were compared between the two groups (immunized and control) using the Mann Whitney U-test and the unpaired t-test. In both cases, the differences were highly significant (P < 0.001), with the new V1 sequences from the control group being more closely related to the challenge stock. To investigate the effect that the nucleotide sequence diversity in V1 had upon the sequences of the envelope protein, this region was translated for each clone. A comparison of the amino acid sequence of part of the V1 region between amino acids 113 and 156 is shown in Fig. 3. The sequences of two of the clones recovered from J150 (Bm4, Bin7) are not included as they coded for a truncated envelope protein. This figure clearly contrasts the sequence of the immunogen (Fig. 3, top line) across the V1 region with those present in the challenge stock (W1 and W3; Fig. 3, lines 2 and 3). Furthermore, it identifies the coding changes that resulted from the differences in nucleotide sequence in clones recovered from control and immunized macaques. One feature of all the new V1 sequences recovered (below the solid line) from immunized macaques were two changes: N to S (amino acid 151) and T to N (amino acid 153). This
869
results in the movement of a potential N-linked glycosylation site from Asn 151 to Asn 153. This sequence change was not detected in the control macaques.
Discussion The identification of the proteins and their epitopes that confer protection is the first step towards the development of effective vaccine strategies against immunodeficiency viruses. To this end, the characterization of virus recovered from immunized infected animals may be useful. This is the first report for immunodefidency viruses where the sequences of the immunogen, the virus challenge stock and the virus that grew in vivo have all been characterized and compared. The aim was to determine what effect, if any, the presence of a preexisting anti-envelope response had made upon the sequence of the env gene. Simian models of infections with HIV and SIV have suggested that immunization with envelope protein alone is sufficient to confer protection (Berman et al., 1990; Hu et al., 1992). In these reports of the successful use of recombinant vaccines, the immunogen was closely related to the virus challenge stock. In the study reported here both T cell and serological responses against the SIV envelope protein were elicited from immunized macaques, including high-titre neutralizing antibodies against the virus challenge stock (W. Jones, P. Silvera & T. Corcoran, personal communications). However, the animals became infected upon challenge. This suggests that none of these immunological criteria correlate with vaccine protection in vivo (Stott et al., 1991). It was possible that sequence variation between the recombinant immunogen and the virus challenge stock was sufficient for failure of this vaccine trial. Nevertheless, sequence differences were observed between virus recovered from immunized infected macaques and that from naive controls. Presumably, these new envelope sequence variants appeared in the immunized animals because they were able to replicate preferentially in the presence of the pre-existing immunity. The location of sequence changes in the env gene, between the immunogen and the virus challenge stock and between the sequences isolated from immunized and control macaques should, therefore, identify epitopes that are important in protection against SIV. A comparison of the immunogen and the virus challenge stock readily distinguishes the V1 region (Fig. 2 and 3) from the V2 to V5 regions (Fig. 1) of SIV gpl20. Over the 365 amino acids encompassing V2 and V5 there are only 12 positions where the amino acid sequence varies and they are scattered throughout the whole of this region of gpl20. In contrast, the V1 region is highly divergent for virus challenge stock sequences at the
870
N . A l m o n d and others
nucleotide level ( > 15 % variation), which translates into a clearly distinct peptide sequence between amino acids 120 and 150. If the positions of sequence variations across V2 and V5 are important in conferring protection, it is likely that they would need to fold to form a conformational determinant. If V1 is important, then both linear and conformational epitopes are likely to be affected by the sequence variation between immunogen and virus challenge stock sequences. The sequence analysis of the env gene recovered from immunized and control macaques in vivo allowed us to distinguish the V1 region from the rest of gpl20. In spite of the presence of anti-envelope antibodies and cellular responses, the sequences across V2 and V5 recovered from immunized infected macaques, as a group, could not be distinguished from those obtained from control macaques. Most sequence variation observed in immunized macaques could be matched with variation in controls, for example variation across the V2 region of macaque J146 is identical to that recovered from control J151. The sole exception was the sequence variation observed in immunized macaque J174 across the V3 equivalent region of SIV gp120. In previous studies of the evolution o f g p l 2 0 from SIVmac251-32H in vivo, this region has remained highly conserved (Almond et al., 1992b). It is of interest to note that on the day of challenge, this animal possessed detectable T cell proliferative responses to this region of the envelope protein (Jones et al., 1992). For HIV-1 the evolution of virus gag gene sequence variants may be correlated with T cell recognition of the specific epitopes in vivo (Phillips et al., 1991). This may be another example of specific T cell reactivity driving the genetic change of immunodeficiency virus sequences, this time the epitope being within the env gene. In contrast, far more sequence heterogeneity was observed in the V1 region of the env gene. V1 sequences identical to those observed in the virus challenge stock were detected in both immunized and control animals. This may be because the sequence analysis was confined to the characterization of proviral sequences of SIV. Specific proviral sequences may survive in the presence of effective antiviral immunity if they remain unexpressed in the host. Alternatively, this may reflect the differences between the V1 sequences of the virus challenge stock and the immunogen. It is the comparison of new V1 sequences that emerged in vivo which distinguished control and immunized macaques. For the control macaques the new V1 sequences that emerged were all closely related to the virus challenge stock sequences, whereas in immunized macaques far more dramatic changes in sequence were observed. The greater degree of genetic diversity observed in new V1 sequences recovered from immunized
macaques was highly significant when assessed by the Mann-Whitney U-test and the unpaired t-test. It is interesting to note that a common feature of all new sequences recovered from immunized macaques were changes at amino acid positions 151 and 153. These changes changes affected the potential glycosylation pattern across the V1 region. Changes in glycosylation have been found to alter the immunogenicity and antigenicity of epitopes in other viral systems (Skehel et al., 1984). More importantly, the sequence across this region of V1 (amino acids 151 to 156) is identical in both the immunogen and all the virus challenge stock sequences characterized. The virus stock used to challenge the macaques is a virus pool containing env gene sequences of considerable heterogeneity (Almond et aI., 1992a). Therefore, it is not possible to determine unequivocally the mechanism by which new viral sequences appear in immunized and control macaques. It may be that all these sequences are present in the virus challenge stock, albeit in very low amounts. Only 16 clones derived from the 11/88 virus challenge stock have been sequenced (Almond et al., 1992a). However, a 15 month longitudinal study of env sequences which appeared in vivo in two macaques inoculated with the 11/88 virus stock did not detect any sequences with an altered pattern of glycosylation across the V1 region of gpl20 (Almond et al., 1992b). It would seem, therefore, that many of the new sequences detected in this study arose from genetic change occurring to virus present in the initial infecting inoculum. To date, there has been little information as to the function of the V1 region o f g p l 2 0 from SIV or HIV. For SIV, it has been observed to be highly variable and heterogeneous in vitro and in vivo for a number of simian models (Burns & Desrosiers, 1991 ; Johnson et al., 1991 ; Overbaugh et al., 1991; Almond et al., 1992a, b). In other viral systems, regions o f sequence hypervariability in structural proteins have been associated with immune escape mechanisms at viral epitopes that are important in protection. The observation that the presence of preexisting anti-envelope immune responses are associated with more rapid sequence variation, specifically in the V 1 region, provides further evidence that this may be an important region of SIV for mediating protection in vivo. So far, other immunological assays of antiviral immune responses have failed to provide an in vitro correlation with protection in vivo. This study, which characterized viruses capable of replicating in an 'immunized' host, provides information for the logical development of improved vaccine preparations against SIV. The authors would like to thank Dr F. Taffs for veterinarysupport, J. Rose for the preparation of DNA from blood and diagnostic PCR, P. Silvera and T. Corcoran for serological data, W. Jones for T cell
S I V env f r o m immunized macaques
data prior to publication and J. Bootman of the NIBSC/MRC PCR Reference Centre for access to reagents and facilities. We also thank Dr E. J. Stott for critical reading of this paper and Miss L. Plumb for secretarial support. This work was supported, in part, by grants from the MRC AIDS Directed Programme.
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