FERTILITY AND STERILITY

VOL. 72, NO. 4, OCTOBER 1999

Copyright 1999 American Society for Reproductive Medicine
Published by Elsevier Science Inc.
Printed on acid-free paper in U.S.A.

The antiviral agents, MAP30 and GAP31,

are not toxic to human spermatozoa and
may be useful in preventing the sexual
transmission of human immunodeficiency
virus type 1
Courtney A. Schreiber, M.D.,* Livia Wan, M.D., Yongtao Sun, M.D., Ph.D.,
Lucy Lu, B.A., Lewis C. Krey, Ph.D., and Sylvia Lee-Huang, Ph.D.
New York University School of Medicine, New York, New York

Received February 1, 1999;

revised and accepted April
20, 1999.
Reprint requests: Sylvia
Lee-Huang, Ph.D.,
Department of
Biochemistry, New York
University School of
Medicine, 550 First
Avenue, New York, New
York (FAX: 212-263-8166).
Supported in part by grant
RO1 AI 31343 from the
National Institutes of
Health, National Institute of
Allergy and Infectious
Diseases, Bethesda,
Maryland (S.L.-H.).
* Present address:
Department of Obstetrics
and Gynecology, Hospital
of the University of
Pennsylvania, Philadelphia.

Department of Obstetrics
and Gynecology.

Department of
Biochemistry.

Laboratory of
Reproductive Biology.
0015-0282/99/$20.00
PII S0015-0282(99)00302-7

686

Objective: To investigate the effects of two virucidal compounds, MAP30 (Momordica anti human immunodeficiency virus [HIV] protein; molecular weight, 30 kd) and GAP31 (Gelonium anti-HIV protein;
molecular weight, 31 kd), obtained from Momordica charantia and Gelonium multiflorum, respectively, on the
motility and vitality of human spermatocytes.
Design: Prospective, controlled study.
Setting: New York University School of Medicine.
Patient(s): Ten healthy men undergoing evaluation for infertility provided 10 semen specimens.
Intervention(s): Human sperm were treated with the anti-HIV agents, MAP30 and GAP31. Nonoxynol-9, a
commonly used spermicide, and phosphate-buffered saline were used as the positive and negative controls,
respectively.
Main Outcome Measure(s): The motility and vitality of human spermatocytes treated with MAP30 and
GAP31 at doses that inhibit HIV-1 and herpes simplex virus.
Result(s): MAP30 and GAP31 did not inhibit the motility or vitality of human sperm cells over a dose range
of 100 0.1 g/mL, whereas nonoxynol-9 demonstrated spermicidal action on all 10 samples over the same
dose range.
Conclusion(s): The antiviral agents, MAP30 and GAP31, were not toxic to human sperm cells at the doses
at which they inhibit HIV-1 and herpes simplex virus. They had no effect on the motility of spermatozoa, even
at a dose of 1,000 times the maximum effective concentration. These results indicate that MAP30 and GAP31
may be useful as nonspermicidal protection against sexually transmitted diseases. (Fertil Steril 1999;72:
686 90. 1999 by American Society for Reproductive Medicine.)
Key Words: AIDS, HIV-1, MAP30, GAP31, nonspermicidal antiviral agents

More than 50% of the pregnancies that

occur in the United States are unplanned, and
the transmission of human immunodeficiency virus type 1 (HIV-1) and other sexually
transmitted diseases (STDs) remains a serious
public health problem. The aim of this study
was to investigate the effects of the antiviral
compounds, MAP30 (Momordica anti-HIV
protein; molecular weight, 30 kd) and GAP31
(Gelonium anti-HIV protein; molecular weight,
31 kd) (1, 2), on human spermatozoa. Our initial hypothesis was that if these compounds
proved to be spermicidal, they could be used as

a topical method for preventing pregnancy and

viral STDs.
However, we found that both MAP30 and
GAP31 had no effect on sperm motility and
vitality at concentrations within the virucidal
range. These results suggest that the compounds may be of value in another arena, that
of achieving pregnancy while preventing
STDs, including HIV infection. The insemination of HIV-negative women with semen from
their HIV-positive partners is becoming more
common. The use of assisted reproductive
technology in HIV-discordant couples has been

tried in Europe with some success. Such treatments primarily

have been based on semen-processing techniques because
immunocytologic studies have shown that HIV-infected
cells are removed from motile sperm by centrifugation and
swim-up separation. Seronegative women who are inseminated with sperm from seropositive men that have been
processed in this manner have a reduced risk of exposure to
HIV-contaminated sperm cells (35).
MAP30 and GAP31 are antiviral agents isolated from
Momordica charantia and Gelonium multiflorum, respectively (1, 2). They exhibit potent inhibition of HIV-1 and
herpes simplex virus (HSV) (12, 6 9). However, MAP30
and GAP31 have no effect on the motility and vitality of
human sperm cells. These findings may offer new, nonspermicidal strategies for preventing the sexual transmission of
HIV-1 and other STDs.

FIGURE 1
Effect of nonoxynol-9 (N-9), MAP30, and GAP31 on the motility of human spermatozoa. The plot shows average values
of triplicate measurements made in duplicate experiments on
10 individual human sperm samples at each drug concentration. All data are expressed as means SEM. The inhibition
of sperm motility by N-9 is dose-dependent. Complete inhibition was achieved at 100 g/mL, whereas MAP30 and
GAP31 demonstrated little inhibition over the entire range of
concentrations tested.

MATERIALS AND METHODS

Semen Samples and Counting Methods
Our experiments were performed with the approval of the
Institutional Review Board of New York University Medical
Center. Semen samples were obtained randomly from 10
healthy donors at the Andrology Laboratory of the Department of Obstetrics and Gynecology at New York University Medical Center. All the specimens were normal semen samples according to World Health Organization
guidelines (10). Each sample consisted of 20 106
motile sperm per milliliter. Sperm count and motility were
determined by placing the sperm in a Mackler chamber
and assessing the samples under a microscopic field at
40 magnification.
Each semen sample was washed twice in Hams F-10
medium. The washed sperm were spun to a pellet and then
resuspended in the medium at a concentration of 40 106
spermatocytes per milliliter with at least 50% motility. Five
microliters of sperm then were suspended in 100 L each of
phosphate-buffered saline and serial 10-fold dilutions of
MAP30, GAP31, and nonoxynol-9 (N-9) ranging from 100
0.1 g/mL. Nonoxynol-9 was used as a positive control.
Motility and forward progression were assessed at each log
concentration of the three compounds. The exposure time
ranged from 5 minutes to 2 hours.

Vital Staining
Vitality staining was performed by adding two drops of
eosin Y to one drop of the sperm sample and mixing for 30
seconds. Two drops of nigrosin then were added to this
mixture and the solution was mixed for 30 seconds. Slides
were made from one drop of each solution mixed with
MAP30, GAP31, or N-9.

Antiviral Compounds
Homogeneous MAP30 and GAP31 were prepared as described earlier (1, 2). These samples were dissolved in sterile
FERTILITY & STERILITY

Schreiber. MAP30 and GAP31. Fertil Steril 1999.

phosphate-buffered saline at a concentration of 1 mg/mL as

stock solutions and kept at 4C until used. Nonoxynol-9 was
purchased from Sigma Chemical Company (St. Louis, MO)
and prepared as a 1-mg/mL stock solution.

Anti-HIV Assay
Activity against HIV was assessed by the inhibition of
viral core protein p24 expression in HIV-infected MT-4 T
lymphocytes by RIA as described previously (10, 11). In
brief, the cells were plated in duplicate in 96-well microtiter
plates at a concentration of 1 105 cells per milliliter in the
presence of serial 10-fold dilutions of MAP30, GAP31, or
N-9 at concentrations ranging from 10 g to 10 pg/mL. The
687

FIGURE 2
Effect of nonoxynol-9 (N-9) and GAP31 on the vitality of human spermatozoa. The sperm samples were stained with vital stain.
Nonoxynol-9, a potent spermicide, exhibits a strong antisperm effect. After treatment with N-9, all the sperm were dead and
permeable to the pink eosin Y dye in the stain. GAP31 was not toxic to the spermatozoa and had little effect on their vitality.
Samples treated with GAP31 remained vital and impermeable to the dye in the stain. Similar results were obtained with MAP30
and with untreated control samples. (Original magnification, 800.)

Schreiber. MAP30 and GAP31. Fertil Steril 1999.

cells then were infected with HIV-1 (IIIB) at 100 infectious

units per cell. The culture supernatants were collected on day
3 and were used for p24 assays performed with an ELISA kit
obtained from Coulter (Hialeah, FL). Duplicates of the original viral inoculum in the same volume of medium without
target cells, incubated for 3 days, were used as blanks. The
blank values were subtracted from wells in which virus was
propagated in host MT-4 cells with or without added drugs.

Anti-HSV Assays
Activity against HSV was assessed with HSV-2 by
ELISA (Meridian Diagnostics, Inc., Cincinnati, OH) as reported previously (6). The human embryonic lung fibroblast
cell line WI-38 (CCL-75; American Culture Type Collection, Rockville, MD) was used as the target cells. Herpes
simplex virus type 2 (VR-734, strain G) derived from an
individual with the genital infection was obtained from
American Culture Type Collection.

RESULTS
Sperm Motility by Counting
The effects of N-9, MAP30, and GAP31 on the motility
of the semen samples obtained from 10 individual donors
over concentration ranges of three logs from 0.1100 g/mL
are summarized in Figure 1. All values are expressed as
percentages of relative motility, using the results from
688

Schreiber et al.

Anti-HIV agents and sperm vitality

samples processed in phosphate-buffered saline as 100%

motility.
At 100 g/mL, N-9 totally inhibited the motility of the
sperm cells from all 10 samples, whereas MAP30 and
GAP31 had only a marginal effect. At 10 g/mL, the maximum effective concentration (EC100) of N-9s antiviral activity, N-9 caused 43% 5% inhibition of motility, whereas
MAP30 and GAP31 caused 4% 4% to 6% 4% inhibition. At lower concentrations of 1 0.01 g/mL (the effective antiviral ranges for MAP30 and GAP31), almost
no inhibition (0%3%) of sperm motility was observed.
However, N-9 exhibited dose-dependent inhibition of
sperm motility.
These results illustrate clearly that in contrast to N-9, the
antiviral agents MAP30 and GAP31 are not toxic to human
spermatozoa. The median effective concentration (EC50) for
the spermicidal activity of N-9 is 20 g/mL and the EC100 is
100 g/mL. These values are consistent with previously
reported results (12). However, MAP30 and GAP31 showed
little spermicidal activity over the entire range of concentrations tested.

Sperm Vitality by Staining

To confirm that MAP30 and GAP31 are not toxic to
human spermatozoa, we performed vitality staining in addition to motility studies by sperm counting. Figure 2 shows
Vol. 72, No. 4, October 1999

FIGURE 3
Effect of nonoxynol-9 (N-9), MAP30, and GAP31 on human
immunodeficiency virus (HIV) as assessed by p24 ELISA of
HIV-infected MT-4 culture supernatant. The plot shows average values of duplicate measurements from three independent experiments. All data are expressed as means SEM.
Complete inhibition of p24 expression by MAP30 and GAP31
was observed at 0.1 g/mL, whereas inhibition by N-9 was
observed at 10 g/mL, approximately 100-fold higher.

TABLE 1
Median and maximum effective concentrations for antiviral
and spermicidal activities of nonoxynol-9, MAP30, and
GAP31.
Anti-HIV
activity*
(g/mL)
Agent
Nonoxynol-9
MAP30
GAP31

AntiHSV-2
activity
(g/mL)

Spermicidal
activity
(g/mL)

EC50

EC100

EC50

EC100

EC50

EC100

0.1000
0.002
0.005

10.0
0.1
0.1

10.000
0.001
0.010

50.00
0.01
0.10

20
No
No

100
No
No

Note: EC50 effective concentration at 50% inhibition; EC100 effective

concentration at 100% inhibition; HIV human immunodeficiency virus;
HSV-2 herpes simplex virus type 2.
* Anti-HIV activity was assessed by inhibition of viral core protein p24
synthesis by ELISA as described in the Materials and Methods section. The
values are averages of duplicates from three independent experiments.
AntiHSV-2 activity was assessed for inhibition of HSV antigen production by ELISA as described in the Materials and Methods section. The
values are averages of duplicates from three independent experiments.
MAP30 and GAP31 also were effective against HSV-1 (4).
Spermicidal activity was measured by inhibition of sperm motility. The
values are averages of three measurements of duplicate experiments at each
concentration in 10 individual sperm samples.
Schreiber. MAP30 and GAP31. Fertil Steril 1999.

GAP31 and MAP30 exerted a similar anti-HIV effect, with

an EC50 and EC100 of 0.001 g/mL and 0.1 g/mL (0.3 nM
and 3.3 nM), respectively. Compared with N-9, which had
an EC50 and EC100 of 0.1 g/mL and 10 g/mL (162 nM
and 16.2 M, respectively, MAP30 and GAP31 were approximately 100-fold more potent in terms of weight per
volume. In terms of molar concentration, GAP31 and
MAP30 were approximately 540-fold more potent than N-9
at the EC50.

Schreiber. MAP30 and GAP31. Fertil Steril 1999.

the typical results of staining. The treatment of human sperm

samples with N-9 had a potent spermicidal effect. The membranes of the dead sperm were permeated by the pink stain.
In contrast, spermatozoa treated with GAP31 and MAP30
remained vital and impermeable to the pink dye.

Antiviral Activity
The anti-HIV activity of MAP30 and GAP31 was compared with that of N-9 by measuring their effect on the
expression of viral core protein p24 expression in HIV-1
infected MT-4 lymphocytes. The results are summarized in
Figure 3. Over a 10,000-fold concentration range, from
0.00110 g/mL (0.03334 nM), MAP30, GAP31, and N-9
all exhibited dose-dependent inhibition of p24 production.
FERTILITY & STERILITY

Comparison of the Antiviral and Spermicidal

Activities of MAP30, GAP31, and N-9
The antiviral activity of MAP30, GAP31, and N-9 and
their comparative spermicidal activity are summarized in
Table 1. The EC50 and EC100 of HIV-1 and HSV infection
were higher for N-9 than for MAP30 or GAP31. Complete
inhibition of HIV-1 and HSV-2 by N-9 was observed at
dosages of 10 g/mL and 50 g/mL, respectively. At these
concentrations, N-9 caused about 50% 80% inhibition of
sperm motility. For MAP30 and GAP31, the EC100 was 0.1
g/mL, for HIV-1, and 0.01 g/mL and 0.1 g/mL, respectively, for HSV-2. These concentrations are 23 orders of
magnitude lower than the EC100 for N-9, and no significant
effect on sperm motility could be detected at these concentrations. It is important to note that these results indicate that
not only are MAP30 and GAP31 more potent than N-9 in
their antiviral action but they also are nonspermicidal.
689

DISCUSSION
In this study, we found that the antiviral agents, MAP30
and GAP31, did not affect the motility and vitality of human
sperm cells obtained from a group of 10 donors. To our
knowledge, this is the first identification of nonspermicidal
agents with activity against HIV.
There is a compelling need to develop vaginal microbicides with antiviral specificity for the prevention of sexual
transmission of HIV and other STDs. Most vaginal virucides
are spermicides that have effects on fertility. These include
N-9 (12), the most common commercial anti-HIV spermicide; the acrosin inhibitor 4-acetamidophenyl 4-guanidinobenzoate (11); gramicidin, the polypeptide antibiotic derived
from Bacillus brevis (13); and gossypol, the polyphenolic
aldehyde extracted from cottonseed (13). Although these
compounds are active against HIV-1, their spermicidal effects limit their use to situations in which conception is not
desired. MAP30 and GAP31 are potent against HIV-1 (13)
and HSV (5), yet they lack spermicidal activity. These
data certainly pique interest about the potential uses of
these proteins in vivo. Options for the prevention of
sexually transmitted diseases are few, and the possibility
of expanding such options renders these compounds worthy of further investigation.
The transmission of HIV-1 has been documented through
artificial insemination with semen from infected donors
(14, 15). Thus, routine HIV-1 screening of all potential semen donors should be performed before artificial insemination (16). Extracellular viral RNA and proviral DNA have
been detected in semen samples obtained from HIV-infected
donors (17, 18). It has been reported that treatment of infected semen samples by gradient centrifugation reduces
extracellular viral RNA and DNA to below detectable ranges
and thus lowers the risk of HIV transmission (19, 20).
Our results suggest that the treatment of semen with
MAP30 or GAP31 alone or in combination with prewashing
before artificial insemination may reduce further the risk of
viral transmission from infected donor to uninfected recipient. These nonspermicidal anti-HIV agents may be useful in
the routine treatment of HIV-1positive semen before IVF.
Couples who are HIV-discordant often desire children to the
extent that they are willing to forego the use of protection
during sexual intercourse. The use of the nonspermicidal
anti-HIV agents MAP30 or GAP31 may offer such couples
additional hope of having healthy infants without infecting the infants mothers. Further studies are needed to
investigate the possible clinical application of these antiviral agents.

690

Schreiber et al.

Anti-HIV agents and sperm vitality

Acknowledgments: The authors thank Xavier Sanchez, B.S., and Erlinda

Macanas, B.S., of the Andrology Laboratory in the Department of Obstetrics and Gynecology at New York University School of Medicine for their
valuable assistance throughout this project. They also thank John Thomas,
M.D., Ph.D., Biochemistry Department, for his help in photographing the
sperm samples.

References
1. Lee-Huang S, Huang PL, Nara PL, Chen HC, Kung HF, Huang P, et al.
MAP 30: a new inhibitor of HIV-1 infection and replication. FEBS Lett
1990;272:12 8.
2. Lee-Huang S, Kung HF, Huang PL, Huang PL, Li BQ, Huang P, et al.
A new class of anti-HIV agents: GAP 31, DAP 30 and DAP 32. FEBS
Lett 1991;291:139 44.
3. Simon M, Fermando M, Ruth A, Rosabel E, Joan H, Javier N, et al.
Human immunodeficiency virus type 1serodiscordant couples can
bear healthy children after undergoing intrauterine insemination. Fertil
Steril 1998;70:359.
4. Semprini A, Levy-Setti P, Bozzo M, Ravizza M, Taglicoretti A, Sulpizio P, et al. Insemination of HIV-negative women with processed
semen of HIV-positive partners. Lancet 1992;340:13179.
5. Semprini A. Reproductive counselling for HIV-discordant couples.
Lancet 1997;349:14012.
6. Bourinbaiar AS, Lee-Huang S. The activity of plant-derived antiretroviral proteins MAP30 and GAP31 against herpes simplex virus infection in vitro. Biochem Biophys Res Commun 1996;219:9239.
7. Huang PL, Chen HC, Kung HF, Huang PL, Huang P, Huang HI, et al.
Anti-HIV plant proteins catalyze topological changes of DNA into
inactive forms. Biofactors 1992;4:37 41.
8. Lee-Huang S, Kung HF, Huang PL, Bourinbaiar AS, Morell JL, Brown
JH, et al. HIV-1 inhibition, DNA binding, RNA binding and ribosome
inactivation activities in the N-terminal segments of the plant anti-HIV
protein GAP 31. Proc Natl Acad Sci USA 1994;91:12208 12.
9. Lee-Huang S, Huang PL, Huang PL, Bourinbaiar AS, Chen HC, Kung
HF. Inhibition of HIV-1 integrase by plant antiviral proteins MAP 30
and GAP 31. Proc Natl Acad Sci USA 1995;92:8818 22.
10. World Health Organization. Laboratory manual for the examination of
human semen and semen-cervical mucus interaction. 3rd ed. New
York: Cambridge University Press, 1993.
11. Bourinbaiar AS, Lee-Huang S. Acrosin inhibitor, 4-acetamidophenyl
4-guanidinobenzoate, an experimental vaginal contraceptive with antiHIV activity. Contraception 1995;51:319 22.
12. Lee CH. In vitro spermicidal tests. Contraception 1996;54:131 47.
13. Bourinbaiar AS, Lee-Huang S. Comparative in vitro study of the
prophylactic effect of contraceptive agents with anti-HIV activity:
gramicidin, nonoxynol-9, and gossypol. Contraception 1994;49:1317.
14. Centers for Disease Control. HIV-1 infection and artificial insemination with processed semen. MMWR Morb Mortal Wkly Rep
1990;39:249 56.
15. Semprini AE, Ravizza M, Muggiasca ML, Giuntelli S, Flore S, Pardi G.
Paternal HIV infection and transfer of HIV from mother to fetus. Br
Med J 1994;308:453.
16. Baccetti B, Benedetto A, Burrini AG. Spermatozoa of patients with
AIDS contain HIV particles. In: Melica F, ed. AIDS and human
reproduction. New York: Karger, 1990:4754.
17. Bagasra O, Farzadegan H, Seshamma T, Oakes JW, Saah A, Pomerantz
RJ. Detection of HIV-1 proviral DNA in sperm from HIV-1 infected
men. AIDS 1994;8:1669 74.
18. Mermin JH, Holodniy M, Katzenstein DA, Merigan TC. Detection of
human immunodeficiency virus DNA and RNA in semen by the polymerase chain reaction. J Infect Dis 1991;164:769 72.
19. Moohan JM, Lindsay KS. Spermatozoa selected by a discontinuous
Percoll density gradient exhibit better motion characteristics, more
hyperactivation, and longer survival than swim-up. Fertil Steril 1995;
64:160 5.
20. Quayle AJ, Xu C, Mayer KH, Anderson DJ. T lymphocytes and
macrophages, but not motile spermatozoa, are a significant source of
HIV in semen. J Infect Dis 1997;176:960 8.

Vol. 72, No. 4, October 1999