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Memórias do Instituto Oswaldo Cruz
Fundação Oswaldo Cruz, Fiocruz
ISSN: 1678-8060 EISSN: 1678-8060
Vol. 91, Num. 3, 1996, pp. 347-348
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Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 91(1), May/June
1996
Immunoreactivity of Brazilian HIV Isolates with Different V3
Motifs
R Michael Hendry/^+, Carl V Hanson, Vera Bongertz^*, Mariza
Morgado^*, Alberto Duarte^**, Jorge Casseb^**, Luis Brigido^***,
Esther Sabino^***, Ricardo Diaz^****, Bernardo Galv o-
Castro^*****
Viral and Rickettsial Disease Laboratory, California Department
of Health Services, 2151 Berkeley Way, Berkeley, CA 94704, USA
^*Departamento de Immunologia, Instituto Oswaldo Cruz, Rio de
Janeiro, RJ, Brasil **Laboratorio de Immunogenetica e Transplante
Experimental, Faculdade de Medicina da Universidade de S o Paulo
***Laboratorio de Retrovirus, Servico de Virologia, Instituto
Adolfo Lutz, S o Paulo, SP, Brasil ****DIPA, Escola Paulista de
Medicina, S o Paulo, SP, Brasil *****Laboratorio Avancado de
Saude Publica, Centro de Pesquisa Goncalo Moniz-FIOCRUZ,
Salvador, BA, Brasil
Code Number: OC96068
Size of Files:
Text: 9K
No associated graphics files
[TABLES AND FIGURES AT END OF TEXT]
A successful HIV vaccine must elicit immune responses which
recognize variants of HIV circulating in the vaccine target
population. It is therefore important to understand HIV genetic
and antigenic variation in geographic areas where vaccine trials
are anticipated.
In 1991, Carrow et al. published the observation that, like
American and African sera, Brazilian HIV^+ sera reacted
extensively with the V3 region of the MN strain of HIV. These
Brazilian sera also readily neutralized HIV-MN (EW Carrow et al.
1991 AIDS Res Human Retrovir 7: 831-837). In 1993, Potts
et al. reported sequence heterogeneity in the V3 region of
Brazilian isolates. Based on overall V3 sequences, the majority
of Brazilian isolates were more similar to those of North America
and Haiti than to African isolates. However, only 59% of
isolates contained proline in the tip of the V3 loop. In this
small study, 40% contained a GPGR motif while 36% contained a
GWGR motif previously reported in some Japanese isolates (KE
Potts et al. 1993 AIDS 7: 1191-1197).
In 1994, Morgado et al. studied isolates from Rio de Janeiro
and S o Paulo, and identified one subtype F isolate and a
putative B/F recombinant. In this study, octameric sequences at
the tip of the Brazilian subtype B sequences were variable and
distinct from those in North America and Europe. The GPGR motif
was found in only 28.5% of the isolates, and GWGR in 43% (M
Morgado et al. 1994 AIDS Res Human Retrovir 10: 569-576).
Divergence throughout the V3 region suggested that Brazilian
isolates might also differ antigenically from prototype subtype
B strains (upon which most experimental HIV vaccines are being
based).
Sequence analysis, and to a lesser extent heteroduplex mobility
analysis, are labor-intensive, and, in any case, possible only
when viral isolates or patient PBMCs are available. We therefore
undertook a study of antibody binding to defined V3 peptides,
using Brazilian sera including many from patients from whom no
sequenced isolates were available.
Sequences of the four peptides used in this study are compared
with that of the B subtype consensus sequence in Fig. BR1
differs from the subtype B consensus only by substitution of "W"
in place of "P" in the GPGR motif. BR2 is a consensus of a
group of Brazilian isolates which possess the GWGR motif. Over-
all, two of the peptides included "P" in the V3 tip motif and two
included "W". The peptides were biotinylated at their amino
terminus and contained a four amino acid spacer. The biotinylated
peptides were captured onto avidin coated microtiter plates.
consB NTRKSIHIGPGRAFY %homology
BR1 .........W..... 93
NR2 ........MW..... 87
SF2 .......Y......H 87
MN .K.R........... 87
Sera were obtained initially from 21 Brazilian patients whose
HIV isolates were of known V3 sequence. Binding of these sera
to the peptides was determined colorimetrically using an enzyme-
labeled anti-human-immunoglobulin and a chromogenic substrate.
Binding was expressed as the reciprocal of the highest antibody
dilution giving an optical density of 0.5. Serum specimens
corresponding to the GWGR motif had highest titers against
the GWGR-containing BR-1 and BR-2 peptides. Specimens
corresponding to isolates with "P" in the motif, had titers that
were more nearly equal against most of the peptides.
Four different ratios of binding titers (BR1-to-SF2, BR1-to-MN,
BR2-to-SF2 and BR2-to-MN) were computed. The sum of these four
ratios would thus be expected to be high for reactivity with the
"W" motif and low for reactivity with the "P" motif. By inspec-
tion of the data, we found that ratio-sums above 16 corresponded
100% with "W" reactivity and ratio-sums below 8 corresponded 100%
with "P" reactivity. Ratios between 8 and 16 were considered
indeterminate. Of five specimens with unusual non-W/non-P mo-
tifs, two typed by this scheme as "W-like", one as "P-like" and
one as indeterminate.
As shown in Table, the ratio-sum technique was applied to sera
from Rio de Janeiro, Bahia and S o Paulo from patients with no
sequenced isolates available. Overall, there were equal numbers
of GWGR and GPGR types. While not statistically significant,
GPGR predominated in the northernmost region (Bahia), GWGR pre-
dominated in the southernmost area (S o Paulo), and there were
equal numbers in Rio de Janeiro, which lies geographically bet-
ween the other two sites. Data for S o Paulo are shown for equal
numbers of samples collected recently and 10 years earlier.
Since there is no significant difference between these, it sug-
gests that GWGR was already common in Brazil early in the epide-
mic. This early, uncommon GWGR strain may thus be a phylogenetic
ancestor of current GWGR strains.
Geometric mean averages of binding titers against each of the
six peptides were computed separately for all P-type and for all
W-type antisera. In the case of peptides BR2, SF2 and MN, titer
differences were especially statistically significant. It is
interesting that titers of P-type specimens were especially high
against the consensus F-type peptide, in contrast to those of W-
type peptides. This is consistent with the "promiscuous", or
relatively broad, reactivity of P-type antisera.
These observations suggest that the choice of a GPGR motif
strain, such as MN, as the basis for a vaccine may be fortuitous
even in an area such as Brazil where multiple HIV subtypes and
V3-motif variants coexist. In the context of vaccine develop-
ment, however, the important issue is the ability of an HIV
antigen to elicit broad functional activities such as virus
neutralization or HIV-specific cytotoxic T-lymphocytes. Neutral-
izations of a Brazilian motif-W virus and a Brazilan motif-P were
therefore studied. For type-W sera, there was a strong selectiv-
ity for neutralization of type-W virus, consistent with the
antibody-binding data. In contrast, neutralization of these same
viruses by type-P antisera overall occurred without preference
for either virus (data not shown).
uIn conclusion, (1) in Brazil there are subtype-B viruses with
different V3 motifs; (2) there are two antigenically distinct
strains of HIV-1 subtype B cocirculating widely in Brazil; (3)
antibody-peptide binding experiments can categorize patients
exposed to HIV variants with different V3 motifs, even in the
absence of sequenced viral isolates; (4) functional antibody
activities, such as virus neutralization, may be correlated with
exposure to different V3 motifs; (5) V3-motif analysis and pep-
tide-binding studies may be relevant to the design of HIV vac-
cines. For example, the GPGR motif of HIV-MN may contribute to
its eliciting a more broadly cross-reactive response than would
be the case with subtype B viruses having other V3 motifs.
^+Corresponding author. Fax: 510-540.3305
Received 7 December 1995
Accepted 10 January 1996
Sequence of the synthetic peptides used.
TABLE
Frequency of serologic reactivity to V3 synthetic peptides in
Brazilian sera
Number (%) of sera
--------------------------------------
Location Date GWGR GPGR Indeterminate Total
----------------------------------------------------------
Bahia 1988-90 11(27) 27(66) 3(7) 41
Rio 1988-92 39(48) 37(46) 5(6) 81
Sao Paulo 1983 18(64) 10(36) 0 28
Sao Paulo 1993-94 15(54) 13(46) 0 28
Total 83(47) 87(49) 8(4) 178
Copyright 1996 Fundacao Oswaldo Cruz
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