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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. 343-345
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Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 91(1),
May/June 1996
Neutralization of Primary HIV-1 Isolated from Individuals
Residing in Rio de Janeiro
V Bongertz/^+, CI Costa, B Grinsztejn*, HEC/FIOCRUZ AIDS
Clinical Research Group^++, JHC Pilotto**, EC Jo o Filho,
MG Morgado ***
Laboratorio de AIDS e Imunologia Molecular, Departamento de
Imunologia, Instituto Oswaldo Cruz, Av. Brasil 4365, 21045-900
Rio de Janeiro, RJ, Brasil *Hospital Evandro Chagas, Rio de
Janeiro, RJ, Brasil **Hospital dos Servidores do Estado, Rio de
Janeiro, RJ, Brasil ***Hospital Geral de Nova Iguacu, Rio de
Janeiro, RJ, Brasil
Code Number: OC96067
Size of Files:
Text: 12K
No associated graphics files
Key words: HIV-1 - primary isolates - neutralization
[TABLES AND FIGURES AT END OF TEXT]
Control of HIV-1 infection has been attempted to achieve by
induction (Karzon et al. 1992 Vaccine 10: 1039-1052) or
passive transfer of HIV-1 neutralizing antibodies (D Vittecoq et
al. 1995 Proc Natl Acad Sci USA 92: 1195-1199, PWHI Parren
et al. 1995 AIDS 9: F1-F6, MC Gaudin et al. 1995 J Inf
Dis 171: 1203- 1209). Evaluation of the efficacy of anti-
HIV/AIDS vaccine candidates has been based on analyses of the
effective humoral immune response presented by neutralizing
antibodies and of the effective cellular immune response
presented by cytotoxic T lymphocytes (KB Cease & JA Berzofski
1994 Annu Rev Immunol 12: 923-89). The difficulty in
inducing an effective immune response against HIV-1 has been
shown to be caused by the extreme variability of this virus, used
by this agent in order to escape immune control (JA Levy 1993
Microbiol Rev 57: 183-263). The initial optimism in
relation to neutralizing antibodies (NAb) has thus decreased
progressively in the last years, mainly after constatation that
the satisfactory neutralization of laboratory HIV-1 isolates
in vitro by patient sera cannot be presumed to indicate
the capacity to neutralize the autologous HIV-1 isolate or the
viral variants which appear during the progression of the
immunodeficiency syndrome (DC Montefiori et al. 1991 Virol
182: 635-643). However, recent data indicate that potent
broadly reactive, protective neutralizing antibodies do exist in
patient sera and can be produced in vitro (JR Mascola et
al. 1994 J Inf Dis 169: 48-54).
Exhaustive efforts aiming to identify HIV-1 variants in
different geographical areas or defined groups of infected
persons have been carried out. Although genotypic typing has
identified different clades of HIV-1, serotyping efforts have not
corresponded at the same level, and definition of main HIV-1
variants through neutralization studies has shown to be extremely
difficult (S Osmanov 1995 personal communication). However,
studies with monoclonal HIV-1 neutralizing antibodies have shown
that some kind of "grouping" of HIV-1 variants according to their
susceptibility to neutralization can be achieved (JP Moore 1994
9eme Coll Cent Gardes 151-155, DR Burton 1994 9eme Coll
Cent Gardes 167-175, EM Fenyoe 1994 9eme Coll Cent
Gardes 103-107, M Robert-Guroff et al. 1994 J Virol
68: 3459-3466, AJ Conley et al. 1994 J Virol 68: 6994-
7000, JP Moore et al. 1995 J Virol 69: 101-109, E
Jurkiewicz et al. 1995 AIDS 9: 91-93).
Several anti-HIV/AIDS vaccine candidates are being evaluated
in diverse clinical trials. Brazil is one of the countries with
some regions in the southeast of the country afflicted with a
very high regional HIV-1 incidence, and the need for vaccines
is urgent. In order to choose vaccine candidates to be applied
in Brazil, basic studies not only of the viral variants
circulating in Brazil but also of the immune response of infected
individuals have to be carried out.
The present study presents results obtained analyzing the
susceptibility to neutralizing antibodies of 27 primary HIV-1
isolates obtained from infected individuals residing in the city
of Rio de Janeiro (Table). Autologous neutralization (defined as
the capacity of antibodies in sera or plasma in neutralizing the
HIV-1 isolate obtained at the same date from the same
individual), heterologous neutralization (susceptibility to
neutralization by NAb in plasma from other HIV-1 individuals),
susceptibility to neutralization by control NAb (sera or pools
of plasma known to neutralize primary HIV-1 isolates) and the
capacity to neutralize a reference HIV-1 isolate (laboratory
strain HIV-1 MN) were determined. Traditional neutralization
assays using a mixture of phytohemagglutinin (PHA) activated
mononuclear cells obtained from HIV-1 seronegative blood donors
were carried out (J Albert et al. 1993 AIDS Res Human Retrovir
9: 501-506).
Results indicated a fairly high percentage of isolates
susceptible to autologous neutralization (11/16 isolates, 68.7%),
when positive autologous neutralization was defined as the
capacity to neutralize more than 50% of the virus in comparison
to viral detection in control wells in absence of antisera.
Preliminary data indicate that this percentage is probably higher
than would be detected in a greater sample (15/26 isolates,
57.7%). When 80% activity reduction is defined as positive
neutralization, 7/16 (43.7%) of the isolates tested were
neutralized by their autologous antisera. Athough no sistematic
titration of NAb was carried out, the great majority of the
autologous plasma neutralized the autologous HIV-1 isolate at
very low titers (1:10 - 1:20). Only four (15.4%) of the isolates
appear to be highly susceptible to the autologous neutralizing
antibodies, as detected by the capacity of autologous NAb to
neutralize higher viral concentrations. Only one (6.7%) of the
primary isolates was completely neutralized by the autologous NAb
(two separate assays).
The majority of the samples with positive autologous NAb were
detected in individuals with a seroconversion period of less than
one year and with higher CD4 positive T cell counts, as reported
in other studies (M Arendrup et al. 1992 J AIDS 5: 305-
307, ML Tsiang et al. 1994 J Inf Dis 170: 1141-1147),
although no statistically significant difference between groups
of patients distributed according to infection time and CD4 T
cell count was found. No correlation to stage of disease, sex,
age or probable mechanism of infection could be detected.
Heterologous neutralization of primary HIV-1 isolates from Rio
de Janeiro was highly variable, as reported in studies from other
geographical areas (WHO Network for HIV Isolation and
Characterization Report). Fifty three out of 75 assays (70.7%)
indicated a greater than 50% heterologous neutralization. Some
HIV-1 primary isolates showed a higher susceptibility (7/21, 33%)
to neutralization, being neutralized by the majority of
heterologous plasma tested (varying from 2/3 to 9/9 assays)
others were quite resistant to heterologous neutralization (2/21,
9.5%), being neutralized by only one of the heterologous antisera
(1/3 to 1/6).
An attempt to analyse difference in susceptibility to
neutralization by plasma from individuals infected with the
Brazilian B' HIV-1 subtype variant or the more
international B variant was carried out but results showed
a qualitative difference for only one isolate. Quantitative
studies may allow distinction.
Susceptibility to pools of plasma from seropositive individuals
was higher when pools of plasma of asymptomatic individuals were
employed (12/12, 100%) than when mixtures of plasma from
individuals in more advanced stages of the disease were used
(17/20, 85%). Susceptibility to specific anti-HIV-1 MN serum was
higher than expected (6/12, 50%).
Neutralization (>50%) of the reference HIV-1 isolate MN was
achieved by 25/28 (89%) of the plasma, confirming results
reported in the literature (V Bongertz et al. 1994 Mem Inst
Oswaldo Cruz 89: 113-114). Although the capacity to
neutralize the HIV-1 MN isolate does not correlate to the
capacity to neutralize either the autologous or heterologous
primary isolates, the reverse situation may indicate a special
lack of capacity to neutralize either autologous or heterologous
isolates: of the three plasma unable to neutralize the HIV-1 MN
isolate, none did neutralize the autologous isolate, and capacity
to neutralize heterologous primary HIV-1 isolates was quite
low.
The neutralization assay described recently (S Zolla-Pazner
1994 9eme Coll Cent Gardes 161-165) as a more sensitive
test for HIV-1 neutralization was also used for determination of
neutralization susceptibility of Brazilian primary HIV-1
isolates. In this assay, the normal human peripheral blood
lymphocytes (PBMC) are not pre-activated by incubation with PHA,
i.e., "resting PBMC" are employed as host cells for HIV-1
infection. Our results indicate that although the tissue culture
infective dose 50% (TCID 50%) of the reference HIV-1 MN isolate
used in our assays was the same in both kinds of assays
(1:62.500), the TCID 50% of the Brazilian primary isolates showed
a reduction (between 2 and 20) of their TCID 50%, preventing
general employment of this assay for evaluation of our isolates
and antisera.
Attempts to correlate clinical data or other data available
(reactivity with synthetic peptides, genotyping results) referent
to the primary isolates/plasma analyzed showed no significant
results. It should be noted, however, that although neither of
the 2 F subtype HIV-1 primary isolates were neutralized by their
autologous antisera, the neutralization of B subtype primary
isolates by sera from individuals infected with the F type
isolates was very high, as well as the neutralization of the F
subtype isolates by sera from individuals infected with B subtype
HIV-1 isolates.
When potency of heterologous neutralization (50%) was compared
to susceptibility to heterologous neutralization (50%) of the
primary isolates, an inverse relationship appeared to exist in
that plasma from individuals infected with an isolate with
greater susceptibility to heterologous neutralization showed a
generally poorer capacity to neutralize other primary HIV-1
isolates. Experiments envolving a larger sample number to allow
statistical analysis are being carried out.
Acknowledgements: to Dr Carlos Bonecker, Instituto Santa
Catarina, Rio de Janeiro, RJ, for donation of blood donor buffy
coats.
Finantial support by WHO/GPA/VDU, UNDP/World Bank, PIAF/Brazilian
Ministry of Health DST/AIDS and CNPq.
^+Corresponding author. Fax: 55-21-280.1589
^++HEC/FIOCRUZ AIDS Clinical Research Group:
S Cavalcante, MCG Galhardo, MRC Guimar es, VC Rolla, VG Veloso
Santos.
Received 7 December 1995
Accepted 10 January 1996
TABLE
Summary of the results obtained in neutralization assays of 10-50
infective units of Brazilian primary isolates or of the reference
HIV-1 MN isolate by antisera or plasma diluted 1:10
Neutralization Extent # neutralized/ %
of N^a / # tested
-----------------------------------------------
Autologous N 50% 11/16 69
80% 7/16 44
Heterologous N 50% 53/75 71
N by plasma pools 50% 12/12 100
17/20 85
N by anti-HIV-1 MN 50% 6/12 50
N of HIV-1 MN 50% 25/28 89
^a: neutralization
Copyright 1996 Fundacao Oswaldo Cruz
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