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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. 335-338
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Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 91(1), May/June
1996
RESEARCH NOTE
A Nationwide Effort to Sistematically Monitor HIV-1 Diversity in
Brazil: Preliminary Results
B Galv o-Castro/^+, JC Couto-Fernandez, MA Mello, MI Linhares-de-
Carvalho*, LR Castello-Branco*/**, V Bongertz**, PCP Ferreira***,
M Morgado**, E Sabino****, A Tanuri*****, the Brazilian Network
for the HIV-1 Isolation and Characterization^++
Laboratorio Avancado de Saude Publica, Centro de Pesquisa Goncalo
Moniz-FIOCRUZ, Rua Valdemar Falc o 121, 40295-001 Salvador, BA,
Brasil
*Ambulatorio da Providencia da Arquidiocese do Rio de Janeiro,
RJ **Departamento de Imunologia, Instituto Oswaldo Cruz, Rio de
Janeiro, RJ^
***Departamento de Microbiologia, Universidade Federal de Minas
Gerais, Belo Horizonte, MG
****Laboratorio de Retrovirologia, Instituto Adolfo Lutz, S o
Paulo, SP *****Laboratorio de Virologia Molecular, Departamento
de Genetica, Universidade Federal do Rio de Janeiro, RJ,
Brasil
Code Number: OC96065
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Key words: HIV-1 - genetic - antigenic - diversity - Brazil
[TABLES AND FIGURES AT END OF TEXT]
The Human Immunodeficiency Virus Type 1 (HIV-1) presents a high
level of genetic variation leading to isolates with divergent
nucleotide and amino acid sequences and distinct biological
properties. This viral diversity is one of the main obstacles for
the development of a universal effective vaccine. Based on HIV-1
env and gag gene sequence data, at least nine
nearly equidistant genetic subtypes belonging to the M (major)
group could be identified. They are designated from A through I
and occurring in different geographic regions of the world (G
Meyers 1994 AIDS Res Hum Retroviruses 10: 1317-1324, G
Kostrikis et al. 1995 J Virol 69: 6122-6130). In addition,
divergent HIV-1 viruses have been identified, and as could not
be classified in any of these subtypes, were recently designated
as group O (outlier) (M Van den Haesevelde et al. 1994 J Virol
68: 1586-1596, LG Gurtler et al. 1994 J Virol 68:
1581-1585, W Janssens et al. 1994 AIDS 10: 877-879).
Thus, to establish a surveillance program to monitor HIV-1
diversity in sites where HIV-1 candidate vaccine will be
evaluated is of paramount importance for the selection of
appropriate vaccine efficacy trials. In this context, the World
Health Organization Global Programme on AIDS (WHO-GPA) organized
a WHO Network for HIV-1 Isolation and Characterization, providing
the basis of global mechanisms for monitoring HIV-1 variability
(WHO Network for HIV-1 Isolation and Characterization 1994
AIDS Res Hum Retroviruses 10: 1327-1343).
Brazil is one of the vaccine trial sites selected by the WHO-
GPA, together with Rwanda, Thailand and Uganda. So far, at least
three different subtypes have been found in Brazil: B, F, and C.
In fact, through an evaluation of 235 Brazilian isolates it was
observed that subtype B was predominant (88.5%) and that only
8.9% and 1.7% of the samples were subtypes F and C, respectively
(K Potts et al. 1993 AIDS 7: 1191-1197, J Louwagie et al.
1993 AIDS 7: 769-780, MG Morgado et al. 1994 AIDS Res
Hum Retroviruses 10: 569-576, JC Couto-Fernandez et al. 1994
AIDS Res Hum Retroviruses 10: 1157-1163, WHO Network for
HIV-1 Isolated and Characterization, 1994 AIDs Rev Hum Retrov
10: 1327-1343, B Galv o-Castro et al. 1995
Actualizaciones en SIDA 3: 173-178, E Sabino et al. 1995
2nd National Conference on Human Retroviruses and Related
Infections, Washington, DC- USA, M Guimar es et al. 1995 1^o
Simposio Brasileiro de Pesquisa Basica em HIV/AIDS abstract no.
17, RJ, Brazil). Interestingly, two samples (0.9%) showed to be
variants resulting from a recombination between subtypes B and
F (E Sabino et al. 1994 J Virol 68: 3640-6346, B Hahn et
al. 1996 J Virol in press).
Low levels of amino acid sequence conservation in the V3 loop
were also seen between the Brazilian sequences and the HIV-1
prototypes currently in use for vaccine development. Indeed, the
comparison with sequences of prevalent North American/European
HIV-1 strains showed that the Brazilian subtype B sequences
present amino acid replacements in some positions giving
distinctive tetrameres at the tip of the V3 loop. In fact, the
GWGR motif at the crown of the V3 loop was detected in 28 out of
71 (39.4%) isolates analyzed, while the highly conserved North
American/European GPGR motif was observed in 25 samples (35.2%).
Also, novel sequences were detected in 18 samples (25.4%) (MG
Morgado 1994 in Processo de desenvolvimento de vacinas anti
HIV/AIDS. Problemas e beneficios PN-DST/AIDS, Brazilian Ministry
of Health). Moreover, the sequencing of the whole gp120 DNA of
one Brazilian isolate with GWGR motif at the tip of the V3 loop
showed an 89.1% homology of nucleic acid sequence with the
prototype HIV-1 B subtype. The highest divergence was found in
the V1-V3 regions (SM Costa et al. 1995 Aids Res Hum
Retroviruses 11: 1243-1245).
In addition, Brazilians infected with HIV-1 presented a lower
specific antibody response against V3 loop peptides of
predominant prototypes of HIV-1 circulating in North America and
Western Europe (EW Carrow et al. 1991 AIDS Res Hum
Retroviruses 7: 831-838, V Bongertz et al. 1994 Braz J Med
Biol Res 27: 1225-1236).
These results suggest that Brazilian HIV-1 strains have genetic
and antigenic differences in comparison with North
American/European prototype strains, which may hamper the success
of immunoprophylatic programmes based on HIV-1 vaccine candidates
currently proposed to be tested in Brazil.
Therefore a Brazilian Network for HIV-1 Isolation and
Characterization (BNHIC) was established in March 1993, as part
of the National Programme of HIV/AIDS Vaccine Development and
Evaluation, nested with Sexual Transmitted Disease/AIDS Programme
of the Brazilian Ministry of Health. This network has similar
organizational structure to the WHO (loc. cit.). Briefly,
BNHIC was organized on a three-tier basis inclunding primary
site, central reference laboratory and secondary laboratories.
Initially the primary cities comprised three previously
selected ones for HIV-1/AIDS vaccine evaluation located in the
cities of Belo Horizonte, MG; Rio de Janeiro, RJ, and S o Paulo,
SP. These sites are responsible for the selection of volunteers,
collection of blood specimens and the shipment of the blood
samples to the Central Reference Laboratory.
The Central Reference Laboratory is the Advanced Laboratory of
Public Health (LASP), Goncalo Moniz Research Center, FIOCRUZ, in
Salvador, BA. This laboratory is responsible for HIV-1 isolation,
expansion and distribution of biological samples and reagents to
the secondary laboratories. The Central Laboratory is also the
HIV isolates national repository and is also responsible for
transferring of technology and traning.
The biological, immunological and genetic characterization of
the specimens is undertaken at the secondary laboratories: (1)
AIDS and Molecular Immunology Laboratory, Department of
Immunology, Oswaldo Cruz Institute, FIOCRUZ; (2) Infectious
Disease Service, Department of Preventive Medicine, Federal
University of Rio de Janeiro; (3) Microbiology and Immunology
Laboratory, Adolfo Lutz Institute; (4) Molecular Virology
Laboratory, Dept. of Genetics, Federal University of Rio de
Janeiro; (5) Retrovirology Laboratory, Adolfo Lutz Institute; (6)
Retrovirology Laboratory, Departament of Virology, Oswaldo Cruz
Institute, FIOCRUZ; (7) Virus Laboratory, Basic Science
Institute, Federal University of Minas Gerais.
The main objectives of the Brazilian National Network are (1)
to develop a system for continuous monitoring the genetic and
antigenic variability of HIV-1 isolates from different geographic
regions of Brazil; (2) to generate basic information of genetic
and antigenic properties of epidemiologically relevant HIV-1
strains that will enable the selection of antigenically
appropriate candidate vaccines to be evaluated and potentially
used in Brazil; (3) to participate in other international HIV-1
characterization efforts as part of the WHO Network for HIV
Isolation and Characterization, or on a bilateral basis of
collaboration with individual international research programs.
This approach, using rigorous standardized research protocols,
will enable a wider ranging analysis of HIV-1 samples from
different regions of Brazil.
In order to achieve these goals, we have been carrying out a
pilot study in one of three previously selected sites, (Rio de
Janeiro, RJ) for future HIV vaccine evaluation.
Initially, 16 seropositive individuals were selected for this
study. HIV-1 isolation and expansion were performed according to
the standard procedures described elsewhere (WHO Guidelines for
Standard HIV Isolation Procedures, Geneva, 1994). Briefly, PBMC
of seropositive and seronegative individuals were separated in
gradient of Ficoll Hypaque, from whole blood collected using
EDTA. 8 x 10^6 PBMC from seronegative donors, previously
stimulated with phytohemaglutinin, were co-cultured with 2 x 10^6
patient cells in RPMI medium containing glutamine, penicilin,
streptomycin and 10% fetal calf serum in the presence of 5U/ml
Interleukin-2. The co-cultures were incubated at 37 C, 5% CO2,
up to 24 days. Culture medium was changed each three to four days
and fresh donor cells were added on the 7th^ and on the 14th
days. Co-cultures were monitored for the presence of the p24
antigen each three to four days and the positive supernatants
were saved as virus stock.
HIV-1 isolates were biologically characterized using the
methodology described elsewhere (WHO loc. cit.). Briefly,
positive supernatants from primary cultures were used as the
virus source to infect MT-2 cell lines. These cultures were
monitored twice a week for p24 antigen and daily for syncitium
formation. The isolates were also analyzed by heteroduplex
mobility assay (HMA) in order to determine the subtypes of HIV-1
(E Delwart et al. 1993 Science 262: 1257-1261). The
HIV-1 neutralization assay was performed following the techniques
described previously (J Albert et al. 1993 AIDS Res Hum
Retroviruses 9: 501-506).
The age, sex, presumed mode of transmission, clinical status
of the patients, as well as results of HIV-1 isolation are shown
in Tables I and II. The majority of the patients were males
(75%). The age ranged from 15 to 61 years. Concerning the
possible transmission route, 50% were male homossexual and/or
bissexual, 43% were heterossexual, and in 6.25% the mode of
transmission was undeterminated. We isolated HIV-1 in 12 out of
16 (75%) samples. The p24 antigen was always detected within 12
days of co-culture. Preliminary results of biologic
characterization show that seven isolates induced syncytium
formation in primary co-culture (PBMC) but only three of them
induced syncytium in MT-2 cell line (Table II). Only one isolate
induced syncytium in MT-2 cell line alone. The rate of virus
isolation and the frequency of syncytium inducing (SI) isolates
were higher than that observed in a previous study (WHO loc.
cit.), which could be due to the variation of the clinical
status of our patients. The genetic analyses of 11 samples using
HMA revealed that 10 were B and 1 was F subtypes confirming
previous studies.
Neutralization assays, carried out according to standard
techniques in pre-activated peripheral blood mononuclear cells
(PBMC) (Albert et al. loc. cit.) indicate that two of
seven isolates tested were neutralized to at least 75% by their
autologous plasma (28.6%) and a third isolate was neutralized to
50%. Six of the seven isolates (85.7%) were neutralized by at
least one heterologous plasma from Brazilian HIV-1 infected
individuals. Of 11 plasma evaluated as to their potency in
neutralizing heterologous primary Brazilian HIV-1 isolates, nine
(82%) were able to neutralize 75% of at least one isolate. All
isolates tested (7/7 = 100%) were susceptible to > 75%
neutralization by a pool of plasma from the patients envolved in
this study. Of 12 plasma tested, nine (75%) were able to
neutralize the reference isolate HIV-1 MN. These preliminary
results indicate that one of the isolates (RJ95006) appears to
be quite resistent to neutralization (autologous and
heterologous), while another isolate (RJ95005) appears to be
highly susceptible to neutralization, with all other isolates
showing intermediate susceptibility. In summary, neutralization
of Brazilian primary HIV-1 isolates appears to be similar to
neutralization of other primary HIV-1 isolates described in the
United States (PD Souza et al. 1995 AIDS 9: 867-874, JP
Moore et al. 1995 J Virol 69: 122-130) and Europe (Eva-
Maria Fen o 1994 personal communication and 9eme col Cent
gardes 103-107).
Some of the individuals enrolled in this pilot study did not
fullfill the inclusion criteria required to participate in this
research project. Most of them were infected for more than two
years, including two cases of AIDS with CD4 cell counts below 200
cells/mm^3. In addition, some of them were under antiviral
treatment. Thus, it is important to point out that the
recruitment of individuals not strictly meeting the inclusion
criteria in this study, such as: (a) be a recent seroconverted
or to have less than two years of infection; (b) have CD4 cell
counts above 200 cells/mm^3 and (c) not be under antiviral
treatment, could jeopardize the information required for future
vaccine efficacy trials.
Nevertheless, this pilot study has demonstrated the feasibility
of the BNHIC, as well as the possibility of the HMA usage for
large scale molecular epidemiological studies in Brazil. This
will enable us to establish a sentinel surveillance on the
prevalence and the dynamic of different HIV-1 genetic subtypes
in various population groups in Brazil.
Finally, we should emphasize that a well organized and
integrated national effort is of paramount importance for
establishing a successful surveillance system to monitor HIV-1
diversity on a nationwide scale.
Acknowledgements: to the techinical assistance of Mrs
Jurema Carrilho.
Partially supported by World Health Organization, Global
Programme on AIDS UNDP/World Bank/Brazilian Ministry of Health
STD/AIDS Program; Programa Institucional de AIDS/FIOCRUZ, Bank
of Brazil Foundation and National Research Council, (CNPq).
+Corresponding author. Fax: 55-71-359.2255
++Dirceu Greco, Paulo Cesar Peregrino Ferreira (UFMG), Jose
Carvalheiro (Ministerio da Saude), Bernardo Galv o-Castro, Jose
Carlos Couto-Fernandez (FIOCRUZ - BA), Frits Sutmoller, Vera
Bongertz, Mariza Morgado, Jose P Simonetti (FIOCRUZ - RJ), Mauro
Schechter, Amilcar Tanuri (UFRJ), Luiz Fernando Brigido de
Macedo, Ester Sabino, Mirthes Ueda (Inst. Adolfo Lutz, SP).
Received 7 December 1995
Accepted 10 January 1996
TABLE I
Epidemiological and clinical data and CD^4 ^+ cell counts from
individuals from Rio de Janeiro, RJ, Brazil
Patients Sex Age PTR^a Clinical status CD4/mm^3
-------------------------------------------------------
RJ001-95 M 35 homo asympt 670
RJ002-95 M 28 bi AIDS 519
RJ003-95 M 15 homo asympt 379
RJ004-95 M 61 ? AIDS <50
RJ005-95 M 58 homo AIDS 191
RJ006-95 M 26 hetero asympt ND
RJ007-95 F 30 hetero asympt 673
RJ008-95 F 42 hetero asympt 473
RJ009-95 F 43 hetero asympt ND
RJ010-95 M 37 hetero asympt 612
RJ011-95 M 49 bi asympt 1098
RJ012-95 M 28 homo asympt 330
RJ013-95 M 45 hetero asympt 1191
RJ014-95 M 53 bi asympt 115
RJ015-95 M 32 bi asympt 549
RJ016-95 F 48 hetero asympt 689
a: presumed transmission route (homo= male homosexual; bi=
male bisexual; hetero= heterosexual)
TABLE II
Genetic and phenotipic characterization of HIV-1 isolates from
individuals from Rio de Janeiro, RJ, Brazil
Neutralization
HIV CPE^a TCID^b Suscept-
Potency^d Sub-
ibility^c types
Patients isola- PBMC MT-2 50% aut het het HMA^e
tion
--------------------------------------------------------------
RJ001-95 Y + + <2 NA NA + B
RJ002-95 Y + - 50 + + + B
RJ003-95 Y + - <2 NA NA + F
RJ004-95 Y + - 750 - + + B
RJ005-95 Y - - 750 + + + B
RJ006-95 Y - + 250 - - + B
RJ007-95 N - - NA^f NA NA ND
B
RJ008-95 N - - NA NA NA ND B
RJ009-95 Y + - 30 - + - B
RJ010-95 Y + + 50 - + - B
RJ011-95 Y - - 10 NA NA + B
RJ012-95 Y + + ND^g ND ND ND
ND
RJ013-95 N - - ND ND ND ND ND
RJ014-95 Y - - ND ND ND ND ND
RJ015-95 Y - - ND ND ND ND ND
RJ016-95 N - - ND ND ND ND ND
a: cytopathic effect (in PHA activated peripheral blood
mononuclear cells and in MT-2 cells), as measured by the presence
or not of multinucleated giant cells (syncitia)
b: TCID = tissue culture infectious dose 50%
c: susceptibility of primary HIV-1 isolates to
neutralization by autologous (aut ) and heterologous (het) sera
from Brazilian HIV-1 infected individuals.
d: potency of the sera from these individuals in
neutralizing heterologous (het) primary Brazilian isolates.
e: heteroduplex mobility assay
f: not available
ug: not done
Copyright 1996 Fundacao Oswaldo Cruz
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