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Antimicrobial Agents and Chemotherapy, April 1999, p. 978-980, Vol. 43, No. 4
Primedica Corporation, Worcester,
Massachusetts1; University of
California, Davis, Davis, California2; and
The Lindsey F. Kimball Research Institute of The New York
Blood Center, New York, New York3
Received 15 June 1998/Returned for modification 18 September
1998/Accepted 19 January 1999
Heterosexual transmission of human immunodeficiency virus type 1 (HIV-1) is the major cause of the ongoing AIDS epidemic. Application of chemical barrier methods is expected to contribute to
the worldwide control of this epidemic. Bovine A safe, effective strategy to
prevent transmission of human immunodeficiency virus type 1 (HIV-1)
would be an important advancement in the worldwide control of the AIDS
epidemic. Eighty percent of new infections occur through heterosexual
transmission in developing countries. Therefore, the selected
prophylactic agents must be cost-effective and stable under a variety
of storage conditions.
3HP- In this study, we used the SIV female rhesus monkey model of sexual
transmission to evaluate the efficacy of 3HP- The study was conducted with eight cycling female rhesus monkeys
enrolled into either a treatment group (six animals) or a control group
(two animals). Control animals were inoculated with virus, to
demonstrate inoculum viability, and not treated with a vehicle. This
study was designed to determine if the formulation (vehicle and active
ingredient) was protective, not to evaluate each component. Adult
female rhesus monkeys were received from the Oregon Regional Primate
Research Center, Beaverton; Laboratory Animal Breeders and Services,
Yemassee, S.C.; or Yerkes Regional Primate Research Center, Atlanta,
Ga. Prior to the study, all animals were tested and determined to be
seronegative for antibodies to SIV, type D retrovirus, and simian
T-cell lymphotrophic virus type 1. All animal care and use procedures
conformed to the revised Public Health Service Policy on the
Humane Care and Use of Laboratory Animals (16a). The
animals were anesthetized with ketamine intramuscularly prior to all
procedures. The SIV stock used in this study contained 105
50% tissue culture infective doses (TCID50) and
3HP- Virus load was determined by limiting dilution coculture of isolated
peripheral blood mononuclear cells (PBMC) with CEMx174 cells
(18). Twelve serial 1:3 dilutions of PBMC, beginning with 106 cells, were cocultured in duplicate with
105 CEMx174 cells per well in 24-well plates. Supernatant
samples were collected after 21 days of culture and stored frozen at
Three of the six treated monkeys (E3G, E2V, and FW3) were negative for
virus recovery by limiting dilution coculture (Fig. 1). Virus was recovered from the
remaining three treated animals, as well as from two control animals
inoculated with a single dose of virus. ELISA for anti-SIV antibodies
confirmed the results of the virus recovery assays; the development
of antibodies in virus-positive animals was observed by 4 to 8 weeks postchallenge (Table 1), while
ELISA values remained below the assay cutoff (optical density, 0.250)
in the virus-negative animals. Control monkeys had recoverable virus by
2 weeks postinoculation; viral loads remained high through week 9.
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Effect of 3-Hydroxyphthaloyl-
-Lactoglobulin on
Vaginal Transmission of Simian Immunodeficiency Virus in
Rhesus Monkeys
ABSTRACT
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Abstract
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References
-lactoglobulin modified by 3-hydroxyphthalic anhydride
(3-hydroxyphthaloyl--lactoglobulin [3HP--LG]) was shown to
inhibit HIV-1, HIV-2, simian immunodeficiency virus (SIV), herpes
simplex virus type 1 and 2, and Chlamydia trachomatis
infection in vitro. Here, we show that 3HP--LG not formulated into
any vehicle protected three of six rhesus monkeys against vaginal
infection by SIV. Incorporation of the compound into an appropriate
vehicle is expected to increase the degree of protection. 3HP--LG
may be effective as a vaginal inhibitor of HIV-1 infection in humans.
TEXT
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Abstract
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References
-LG, a form of bovine -lactoglobulin (-LG) (a protein
present in whey and milk) modified by 3-hydroxyphthalic anhydride (3HP), has been shown to inhibit HIV-1, HIV-2, simian
immunodeficiency virus (SIV), herpes simplex virus type 1 and 2 (HSV-1 and HSV-2), and Chlamydia trachomatis infection in
vitro (3, 4, 14-16). Against HIV-1, 3HP--LG has been
shown to be a more potent antiviral inhibitor than sulfated
polysaccharides; it appears both to be virucidal and to block virus
attachment to the CD4 receptor (4, 13, 15). 3HP--LG has
been demonstrated to have broad-spectrum activity against various HIV-1
clades and against syncytium- and non-syncytium-inducing viruses
(14, 15). It does not appear to be affected by exposure to
elevated temperatures (13, 14).
-LG in preventing vaginal infection with cell-free SIV. Although the exact mode of HIV-1
transmission across the vaginal mucosa is not well understood, it is
likely that cell-free virus plays a major role. SIV infection of rhesus
monkeys causes an acquired immunodeficiency disease with a striking
similarity to HIV-1 and HIV-2 infections in humans and is therefore a
relevant model for HIV-1 infection of humans (6).
Genetically, the virus has the same auxiliary gene structure as HIV-2
and shows similar modes of infection, cellular and tissue tropism, and
clinical disease course. The transmission of cell-free virus across the
vaginal mucosa has been well described (1, 2, 7, 17), and
the rhesus monkey model has been used to test the efficacy of nonoxynol
9 in the prevention of infection. Here, we describe the successful
prevention of infection in 50% of the rhesus monkeys that were treated
intravaginally with 3HP--LG.
4.3 × 109 SIV RNA copies per ml. This stock has
been shown to infect 19 of 19 females after a single intravaginal
inoculation (6a, 12a). The animals were treated and
challenged on day 1 of the study and then were monitored by virus
recovery and antibody assays for seroconversion for at least 28 weeks.
-LG (60 mg/ml in phosphate-buffered saline [PBS]
[13]) and virus were inoculated with a 1.0-ml syringe
as previously described (9). 3HP--LG (30 mg in 0.5 ml)
was administered approximately 10 min prior to and 60 min after a
single virus inoculation. The animals were monitored for approximately
6 months postchallenge.
70°C until analyzed for p27 antigen with a p27 antigen assay kit
(Coulter Immunology, Hialeah, Fla.). Serum was analyzed for anti-SIV
antibodies by using a whole-virus enzyme-linked immunosorbent assay
(ELISA) as described previously (18).
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FIG. 1.
Cell-associated viral loads. The numbers of PBMC needed
to recover SIV were quantitated by threefold limiting dilution
coculture. Control animals and treated animals that were virus positive
were not monitored past 4 or 8 weeks. Virus-negative animals were
assigned values of 0, 0.5, and 0.7 to facilitate interpreting the
graph. 3HP, 3HP- -LG.
TABLE 1.
Anti-SIV antibody responses
of monkeysa
A similar degree of protection has been obtained with two forms of
nonoxynol 9, foam and gel (8), where three of six and two of
six animals, respectively, were protected against vaginal transmission
of SIV. One difference between the present study and the nonoxynol 9 study is that 3HP--LG was not formulated in any vehicle gel or foam
but only suspended in PBS. Therefore, it appears that the level of
protection reported here must be due to 3HP--LG or PBS. The
protective efficacy of 3HP--LG against vaginal infection of mice by
HSV-2 was greatly enhanced by its incorporation into a gel formulation
(5). Similar enhancement of the antiviral effect of
3HP--LG would be expected in the SIV-monkey model.
The significance of this data in predicting the effect of 3HP--LG on
HIV-1 transmission in humans should be evaluated in the context of the
model and the potential epidemiologic impact of this level of
protection. The infection of rhesus monkeys with SIV across mucosal
surfaces has been extensively studied (1, 2, 7, 17); the
localization of virus-infected cells, as well as the reproductive tract
pathology in SIV-infected monkeys of both sexes, is similar to
that in humans (11, 12). In order to ensure a 100%
infection rate in control animals by vaginal inoculation, a dose of
105 TCID50 is utilized in this model, a
dose that contains approximately 4.3 × 109 RNA
copies per ml. In human semen, RNA copy numbers as high as 1.3 × 107 per ml have been observed. This indicates that our SIV
challenge uses a dose at least 100 times that likely to occur
during natural exposure. There is no direct correlation between
RNA copy number and infectious titer. Thus, direct comparisons of
RNA copy number in the SIV stock and semen samples are only crude estimates.
The infectivity of a vaginal viral inoculation is likely to be related to some inherent properties of a particular virus strain; the properties which would favor transmission are not well understood (19) but appear to be related to in vivo replication capacity (12a). In our model, although the exact animal infectious dose of virus is not known, virus stocks with TCID50 of 103 and 104 do not reliably infect rhesus monkeys after one vaginal inoculation (6a). Therefore, it is likely that a best estimation of the virus inoculum used would be between 1 and 10 animal infectious doses.
3HP--LG treatment resulted in protection of 50% of the animals
under challenge conditions that exceed natural exposure in humans but
are within reasonable dosage ranges for other animal protection models.
3HP--LG has been shown in vitro to have broad specificity across
clades and is an abundant, cost-effective by-product of the dairy
industry. In addition, 3HP--LG application does not appear to result
in any irritation to mucosal tissues (12b), a problem that
has been observed with nonoxynol 9 and one that theoretically could
promote virus transmission due to vaginal inflammation. The data
presented here suggests that 3HP--LG may play a significant role as
a vaginal inhibitor of HIV-1 infection in humans.
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ACKNOWLEDGMENTS |
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This study was supported by Dairy Management, Inc., Rosemont, Ill.
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FOOTNOTES |
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* Corresponding author. Mailing address: Primedica Corporation, 57 Union St., Worcester, MA 01608. Phone: (508) 890-0100. Fax: (508) 753-1834. E-mail: michael.wyand{at}primedica.com.
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Antimicrobial Agents and Chemotherapy, April 1999, p. 978-980, Vol. 43, No. 4
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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