AIDSWEEKLY Plus; Monday, May 4, 1998
Daniel J. DeNoon, Senior Editor

It is hoped that identification and purification or synthesis of the factor will result in an extraordinarily effective AIDS therapy.

Robert C. Gallo and colleagues at the Institute for Human Virology, who were the first to report that hCG could reduce KS lesions, now call the putative peptide hCG-associated factor or HAF.

They have shown that HAF has dramatic anti-HIV, anti-SIV, and anti-KS effects in animal models. Similar effects were seen in some patients treated with crude HAF preparations, but not in others.

"We anticipate that responses more consistent with those observed in the animal model will be achieved when the active fraction is completely purified and administered by the best route and at proper doses," wrote Yanto Lunardi-Iskandar, Gallo, and colleagues. "Identification of the active moiety(ies) of HAF(s), its large scale production, elucidation of the mechanism(s) by which it exerts its beneficial effects, and ultimately clinical trials with the purified factor(s) are high priorities."

Lunardi-Iskandar et al. reported their findings in the journal Nature Medicine ("Effects of a Urinary Factor from Women in Early Pregnancy on HIV-1, SIV, and Associated Disease," Nature Med, 1998;4(4):428-34).

An earlier report, based on a Gallo presentation to the 1997 Strategic Program for Innovative Research on AIDS Treatment (SPIRAT)/National Cooperative Drug Discovery Groups for the Treatment of HIV Infection (NCDDG-HIV) meeting, appeared in AIDS Weekly Plus, July 7, 1997.

At that time, Gallo and colleagues believed that HAF was the natural urinary proteolytic product of the hCG (beta) chain (the hCG (beta)-core).

"Our present results show that this is wrong," Lunardi-Iskandar et al. wrote. "There is no activity at all with the (beta)-core purified to homogeneity."

The new findings may put to rest a controversy that has dogged researchers since the first demonstration of hCG's anti-KS effects. Some researchers were able to reproduce the anti-HIV/anti-KS effects, and some were not.

The new studies show that active HAF is associated with, but not identical to, hCG. HAF can be found in some commercial hCG preparations (HAF-C) and in urine concentrates from women in the first trimester of pregnancy (HAF-U).

HAF activity could be traced to two fractions: fraction #65 (15- 30 kDa) and fraction #76 (2-4 kDa).

"The active moieties have the characteristics of proteins or peptides based on their susceptibility to protease digestion and heat activation," Lunardi-Iskandar et al. wrote. "These findings also confirm that the active moiety is not the (beta)- or (alpha)-chains, or the (beta)-core of hCG, but is either a different molecular species which co-purifies with some preparations of the hCG and the (beta)- chain, or a breakdown product of the (beta)-chain, and that the variable activity of different clinical-grade preparations [of hCG] is due to differences in their source, their methods of preparation, or both."

The researchers were unable to prove that the two active fractions are derived from the same molecule or molecules or that they are related to one another.

Interestingly, the researchers created synthetic hCG (beta)-chain peptides, which they dubbed satellins. The satellins had the same properties as the active moieties of HAF, they reported, "but at a much lower specific activity."

Researchers at Gallo's lab were the first to show that hCG is active against Kaposi's sarcoma (KS). The finding was accidental, or, as Gallo put it, "a mistake with unconscious intention."

Male and female nude mice were housed together shortly before transplantation with KS-derived KSY-1 cells. The cells induce metastatic cancer in the immunodeficient nude mice, but some mice surprisingly did not develop tumors. It turned out that they were pregnant.

Later studies showed that sera obtained from murine or human females in the early stages of pregnancy could inhibit KS; the anti-KS factor was associated with presence of hCG.

University of Southern California researcher Parkash S. Gill and colleagues, working in collaboration with Gallo, reported at the 1996 Retrovirus conference that intralesional injections of hCG dramatically reduced KS lesions in AIDS patients. Later studies showed that KS lesions vanished in 10 of 12 subjects who received the highest doses of the most effective hCG preparation.

Gallo said that hCG obtained very early in pregnancy is most effective. Since commercial hCG preparations are pooled, efficacy varies from manufacturer to manufacturer and even from lot to lot. Lunardi-Iskandar et al. showed that the most active clinical grade crude hCG preparation was hCG-APL (Wyeth-Ayerst). Also active were CG10 (Sigma) and hCG-Pregnyl (Organon); least active were Goldline (Steris) and Profasi (Serono). None of the purified hCG preparations tested had any HAF activity.

The direct anti-HIV effects of maternal hormones were first reported by Abner Notkins of the U.S. National Institute of Dental Research (De, S.K. et al., J Clin Invest, 1997;99(7):1484-91). Notkins was studying transgenic mice engineered to contain an incomplete set of HIV genes in the DNA of every cell in their bodies. The virus could not reproduce, but could trick certain mouse cells into making many of the viral proteins. Baby mice that inherit HIV DNA from each transgenic parent eventually accumulate enough viral protein that they develop skin lesions, wasting syndrome, and die within three to six weeks.

Notkins and colleagues first recognized that maternal hormones might have a protective effect against HIV when they observed that newborn transgenic mice appear normal in size and weight, but soon show marked growth retardation.

By eight days of age, they weighed 60 percent less that their normal littermates. It appeared to the investigators that the baby mice were protected by maternal hormones during pregnancy, and became susceptible to the effects of HIV once the level of hormones dropped dramatically at birth.

Further experiments showed that when the mice were treated with hCG, the majority of the animals showed normal weight gain as long as they were receiving the hormone. Furthermore, their cells produced considerably less viral protein than animals not receiving hormone treatment, and skin lesions were reduced dramatically.

Lunardi-Iskandar et al. performed a similar set of experiments in transgenic mice.

"Although [Notkins and colleagues] concluded that the effect was due to hCG, we have proven here that it is not hCG," they wrote.

The transgenic mouse experiments indicated that the anti-HIV effect of HAF may be interference or inhibition of viral RNA expression or destabilization of mRNA. In vitro studies, however, showed that HAF had a greater anti-HIV effect than mere inhibition or HIV production from chronically infected cells. This observation suggests additional mechanisms of action.

The anti-HIV effects were not strain specific. Similar HAF antiviral activity was seen with eight strains of HIV-1. Four of these strains were laboratory strains, two were isolates from Nigerian AIDS patients passaged once in a T-cell line; and two were unpassaged primary isolates from Trinidadian AIDS patients.

"No difference was found with the different target cells or type of HIV," Lunardi-Iskandar et al. noted.

This work was supported in part by NIH grant HD 15454.

The corresponding author for this study is Robert C. Gallo, Institute of Human Virology, University of Maryland, 725 West Lombard Street, Baltimore, Maryland 21201-1192.

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