ADVANCE IN THE BATTLES AGAINST HIV

Yinhuai Chen
Ben Liu
Joseph Messer
Va Lip

INTRODUCTION

Acquired Immunodeficiency Syndrome (AIDS) has become a world wide epidemic. AIDS is caused by an enveloped retrovirus containing ssRNA, which is known as the Human Immunodeficiency Virus (HIV). The infection begins when the virus binds its envelope glycoprotein gp120 to the CD4 molecule of the host cells. With its viral reverse transcriptase the virus is able to copy its RNA into DNA. The DNA is incorporated into the host chromosomal DNA forming a provirus. The provirus can remain in a latent state for various periods of time. As a damaging result HIV impairs the function of the immune system by destroying the CD4+ T-cell and the CD4+ TH-cell. The development of treatment against HIV has not been very successful. Over the past decade, various anti-HIV therapies have been tested, but none of them has been able to eliminate the virus. However, recent advances in research and clinical trials for HIV treatment involving anti-HIV drugs, gene therapies and vaccines shed lights that HIV could be effectively controlled and probably will be eventually eliminated.

ANTI-HIV DRUGS

The anti-HIV agents intervene different points in the replication cycle of HIV. They include inhibitors of viral attachment, reverse transcription, protease, TAT regulatory protein, and an increasing array of novel agents directed at other viral targets. Those approved for use in patients include Zidovudine (AZT), Didanosine(ddI), Zalcitabine(ddC), Stavudine(D4T), Lamivudine(3TC), Saquinavir, Ritonavir, Indinavir, and Nevirapine [1]. These agents do not completely inhibit HIV replication, and their duration of clinical benefit has been limited. Side effects are also often observed. The following is several examples of recent development.

  1. New saquinavir dramatically reduces viral load in blood.

Saquinavir is a protease inhibitor. It is enormously powerful in the test tube, but is broken down so efficiently in the human body that only about 4% of the drug swallowed actually gets used. Roche, a pharmaceutical company has developed a new, improved saquinavir in a soft gelatin capsule. Roche says its bioavailability is 3-4 times higher than the currently available hard-gel capsule. [2]

John Gill and colleagues in Southern Alberta HIV Clinic, Calgary, Canada conducted a 24-week open-label study of saquinavir soft-gel capsules in 442 volunteers (10% female, 27% non-white, 95.5% antiretroviral experienced, 18% had previously used a protease inhibitor). Before starting the study, at least half the group had a CD4 count of 201 cells or higher and a viral load of 17,783 copies/ml. After 24 weeks, 43% of participants had viral loads below 400 copies/ml. [3]

The son-of-saquinavir seems to compare favorably to the current market leader, indinavir. On November 7, 1997, based on the results of clinical trials, FDA approved the new soft-gel formulation of saquinavir, which will be sold under the brand name of Fortovase. [2]

  1. DMP-266 -- new drug looks good

DMP-266, also called efavirenz, is a non-nucleoside reverse transcriptase inhibitor, a member of the same class of anti-HIV drugs as nevirapine and delavirdine. The most attractive feature of efavirenz is its once daily dosing.

Mayers and colleagues conducted a 24 week study of indinavir and efavirenz. 101 HIV-positive volunteers were randomly assigned to receive 800 mg indinavir three times daily plus either 200 mg efavirenz taken once daily or a placebo. Before starting the study drugs, the volunteers had an average CD4 count of 283 cells and average viral load of 109,648 copies/ml. After 48 weeks, the indinavir/d4T group had a drop of at least 1.89 logs, with 68% of the group having fewer than 400 copies/ml, and an increase of 150 CD4 cells. The indinavir/d4T/efavirenz group had a drop of 2.38 logs with 88% below 400 copies/ml, and an increase of 245 CD4 cells. [4]

  1. Cancer drug shows considerable promise against HIV

The anti-cancer drug Hydrea (hydroxyurea) has anti-HIV activity when used by itself in lab experiments. When used together with the antiretroviral ddI, it boosts levels of ddI inside cells, thereby increasing its anti-HIV effect. Most recently, in a study of 21 people pre-treated with or intolerant to AZT, the combination ddI/Hydrea was able to reduce blood levels of HIV to 1/10th their pre-study level. The decrease, however, lasted for only 12 to 16 weeks. However, reports from Germany and France now suggest that when used with ddI in at least 3 patients, Hydrea appeared to suppress HIV long after treatment was stopped. The amount of HIV in their blood fell below the level of detection and remained that way for the two years. As well, viral load was also undetectable in lymph node samples taken from the patients. [2]

  1. You got to have HAART

Highly active antiretroviral therapy, or HAART, is often taken to mean a triple or quadruple combination of anti-HIV drugs, featuring at least one protease inhibitor. Multiple agents might provide additive or synergistic activity against the virus. Even more important is the impact of combination therapy with agents requiring different simultaneous mutations for acquisition of viral resistance. The first major advance in this area has been the recent approval (September 29, 1997) of the drug Combivir (combination of 3TC and AZT) by FDA [2]. In clinical trials, however, combinations with more anti-HIV drugs have been used. Researchers in Germany tested a triple therapy--loviride with 3TC and AZT and found it much more effective than the double combination of loviride with AZT in term of the amount of HIV in the blood [5]. A cocktail of 5 anti-HIV drugs including AZT, ddC, DDI, 3TC and interferon-alpha was applied to 6 HIV-infected subjects in a recent study by Saget, et al. [6]. The viral load fell to only 1/1000 its pre-treatment level! One subject had a normal CD4+ cell count and the amount of HIV RNA in his blood was less than 12 copies/ml.

GENE THERAPIES

The increasing data have shown that no any drug can suppress HIV in a long term [7]. Gene therapy, which confers long-term activity effects, may be the most promising therapeutic option against HIV. The choice of antiviral genes and the choice of gene delivery vehicles are the key determinations of gene therapy.

  1. Are there AIDS - resistance genes in human bodies?

It has been found for many years that some individuals can escape HIV infection despite being at high risk for it and other people infected with HIV progress to AIDS slowly. The important findings in the last year explained the phenomena. In addition to CD4+ receptors on both macrophages and T cells chemokine receptors are also essential for HIV entry into those cells [8]. HIVs can be categorized into two group based their ability to infect distinct target populations [8, 9], macrophage-tropic (M-tropic) viruses, which invade macrophage by binding (through their gp120 proteins) to the molecules CD4 and CCR5 ( chemokine receptor) on the macrophage surface; T cell line-tropic (T-tropic) viruses, whose gp120 molecules can recognize CXCR4 (chemokine receptor) protein on CD4+ T cells. O'Briens group found that there were two CCR5 alleles in humane, the less common one has 32 nucleotides missed [9]. Individuals who had homozygous mutant CCR5 alleles could escape HIV infection. People with single deletion mutant of CCR5 allele progressed to AIDS more slowly than those with two normal CCR5 alleles. The mutant CCR5 allele was called "AIDS-resistance gene". In fact, other chemokine receptors, such as CCR-3 and CCR2b, can also serve as coreceptors for HIV [8, 9]. Based on those discoveries new strategies of gene therapy have been proposed [9], which focus on searching for the ways to inhibit HIV binding to CCR5. One strategy is to provide macrophage with engineered genes which can suppress CCR5 expression or block the HIV binding site on CCR5. To AIDS patients, after their HIV-infected blood cells are destroyed bone marrow with homozygous mutant CCR5 genes can be transplanted to rescue their blood cells.

  1. How to destroy HIV RNA and eliminate HIV from infected cells?

HIV is an RNA virus that can exist as a provirus (integrated form on the chromosome of infected cells). Continuing and high-level replication of HIV results in AIDS. Inhibition of HIV replication will delay or prevent AIDS development. Ribozymes are small catalytic antisense RNAs that bind and cleave specific sites in target RNAs. Ribozymes targeting to HIV RNA have been widely investigated and have demonstrated the ability to suppress HIV replication in different cell lines. [10, 11, 12, 13]. The clinical aspects of ribozymes as therapeutics in gene therapy have been evaluated [14]. The "first trial" of a gene therapy against HIV infection by ribozyme has been undertaken in Australia [15]. A new strategy which combines the application of intracellularly expressed anti-Rev (HIV regulatory protein) fragment and a ribozyme specifically targeting the Rev response element has been developed and has shown the potence to block HIV replication [16]. In addition, a hypothetical technique has been proposed for the elimination of all the integrated HIV provirus from infected cells [17]. In this strategy HIV genes will be excised through homologous recombination, resulting in the replacement of HIV proviral genome with therapeutic DNA.

  1. How to deliver anti-HIV genes and obtain long-term gene expression?

Construction of effective vectors for delivery and long-term expression of anti-viral genes is the key point in gene therapy. Antiviral genes are usually transferred into CD4+ T cells or CD34+ progenitor cells by retrovirus in vitro. These cells with engineered genes are then infused into autologous or syngeneic / allogeneic recipients. That approach was thought to be impractical [13]. Due to the ability of HIV to specifically target CD4+ cells and non-cycling cells, HIV vectors have been developed and were thought to be promising vectors for in vivo gene transfer against HIV infection [13]. In fact, HIV may be "a preferred vector system" among the retroviral family - lentiviruses [18]. Most recently, a chimeric viral vector system that combines the high-efficiency in vivo gene delivery characteristics of recombinant adenoviral vectors with integrative capacities of retroviruses has been established and may allow the delivery and long-term expression of antiviral genes [19].

VACCINES

There are two main classes of vaccines in development against HIV today. The first of these two classes is a therapeutic vaccine which is to, "Provoke an immune response in antibodies and cells in order to suppress HIV infection and halt disease progression" [20]. The second class of vaccine is preventative, which is to protect an individual from HIV infection.

  1. Can a vaccine help someone who is already infected?

Yes. Today there is only one vaccine in use as a therapeutic vaccine and that is the rabbis virus vaccine [20]. This vaccine produces an immune response which helps the bodies own immune system rid itself of the virus and the appearance of symptoms. Following this model there are three possible vaccines in testing today, these are gp 120, gp 160, and manisyl.

Manisyl is a retrovirus (BIV) which is related to HIV but is not pathogenic to humans, but can this virus can stimulate an immune response to cross react with HIV. This vaccine creates an immune response to a protein antigen, which is similar to that of HIV. "This protein, p-26, induces the production of a cytotoxic cd8+ cell response which destroys cd4+ cells which are infected with HIV and allows continued surveillance and destruction of any new cd4+cells which may become infected with HIV overtime" [21]. The initial response to Manisyl is a drop in the cd4+ cell count which accounts for the destruction of the HIV infected cd4+ cells. Next there is a rise in the concentration of cd4+ cells in ones serum and this elevation is maintained for a long period of time.

The initial data seems promising. Manisyl is said to stimulate both specific and nonspecific immunity. "Manisyl [can] possibly exerts its positive effects by restoring the bodies capacity to respond with useful antibodies and killer t-cells" [21].

The next two vaccines are gp 120 and gp 160. Both of these vaccines recognize outermembrane proteins of the HIV virus. The vaccine gp 160 has been used inmost phase one testing phase one testing and has shown some promise.

During a phase one trial, 21 asymptomatic patients who were HIV positive were given the gp 160 vaccine. Each one of the test subjects had less than 500 t-cells per ml in their blood. Christos Tsoukos in Canada performed this experiment. The results showed that,"90% developed either new or augmented antibody responses to specific gp 160 epitopes and had a significant rise in t-cell counts." [20]. [the data for this was not shown] Another experiment, by Dr. Smitri Kundu, showed that gp 160 injections were able to stimulate a lymphocyte which could destroy a HIV infected cell [20].

The vaccine gp 120 was shown to induce cellular and humoral responses by Deborah Bifx at the Walter Reid Hospital. Yet there were some side effects including an increase in liver enzymes in the blood.

  1. Is there research being done to find a vaccine to protect those who are not infected with HIV and protect them from infection?

There are many different theories of what a preventative HIV vaccine should contain. This ranges from a vaccine for a specific viral protein to a live HIV virus. One of the most promising vaccines binds to rgp 120. There are two forms of rgp 120. The first has been shown to provide chimps with immunity against HIV. This vaccine was given intravenously with an alum adjuvant. "Under optimal conditions (i.e., high circulating neutralizing and v3 loop antibodies titers), recombinant HIV gp 120 candidate vaccines have protected chimps from chronic infection after challenge with intravenous free or cell-associated live, homologous HIV" [22]. Also, "Chimps immunized multiple times with gp 120 from either the SF2 or MN strains of HIV have been protected against challenge with HIVSF2 grown in human peripheral blood mononuclear cells" [22]. The chimps immune system produced antibodies which could neutralize the HIV virus when given intravenously into the body. This immunity has lasted over two years now.

The second form of rgp 120 is immuno rgp120, which is produced in mammal cells and contains a more glycosylated than the previous rgp 120, which was made in insects. The results of this vaccine study showed that after three injections volunteers produced antibodies which could bind rgp 120 and stimulate a cellular host immune response.

Another preventative vaccine in testing now is gp 120. this vaccine was produced by Dr. James Kahn of the Univ. of San Fransisco. This vaccine was made from the SF2 strain of HIV and is fully glycosylated. This vaccine mimics the original HIV outermembrane protein. The antibody can also bind the cd4 receptor of HIV. [20]

The final theory which has been presented by Dr. Jonas Salk, the creator of the polio vaccine. He suggests that an effective vaccine against HIV must stimulate the cellular response and not the humoral response. (this was presented in the Amsterdam International Conference) His suggestion is that a "miniscule amount of the virus can be able to elicit a cellular response without eliciting antibodies" [20]. This procedure will stimulate an immune response and protect an individual from HIV infection.

REFERENCES

  1. Threlkeld SC, & Hirsch SM. 1996. Medical Clinics of North America 80: 1263-1282
  2. http://www.catie.ca/aids. (a HIV/AIDS treatment information network, updated daily)
  3. Gill MJ, Beall G, Beattie D, et al. [Abstract I-90]. 37th ICAAC, Toronto, 1997.
  4. Mayers D, Riddler S, Bach M, et al. [Abstract I-175]. 37th ICAAC, September 1997.)
  5. Staszewski S, Miller V, Rehmet S, et al. 1996. AIDS 10: F1-F7
  6. Saget BM, Elbiek T, Guthries J, et al. 1997. We. B. 533
  7. Cohen J (1997). Science, Vol. 277, 32-33
  8. Harden, V. & D souza, P. www.critpath.org (website for Critical Path AIDS Project founded by persons with AIDS to provide treatment, resource, and prevention information in wide-ranging levels of detail)
  9. Obrien S.J. & Dean, M. (1997). Sci.Am., 277 (3): 44-51
  10. Sun LQ., Ely, J. A., et al (1997). Mol. Biotechnol. 7(3): 241-251
  11. Duarte, E. A., et al (1997). Methods Mol. Boil. 74:459-468
  12. Cagnon, L. & Rossi, J. (1997). Methods Mol. Biol. 74:451-457
  13. Poeschla, E. et al (1996). Proc. Natl. Acad. Sci. USA, Vol 93: 11395 -11399
  14. Looney, D. & Yu, M. (1997). Methods Mol. Biol. 74:469-486
  15. News: " First trial of anti-HIV gene therapy" (1997). Nature, 388 (6642): 510
  16. Duan, L, et al (1997). Gene Ther. 4(6): 533-543
  17. Batchu, R.B. & Hinds, T. (1997). Med. Hypotheses, 49 (1): 35-39
  18. Janks, S (1997). J. Natl. Cancer Inst. 89(16): 1182-1184
  19. Bilbao, G., et al. (1997). FASEB J. 11:624-634
  20. www.aegis.com (aids education global information system, including a breif description of different aids vaccines in reasearch now.)
  21. www.aidsvaccine.com (This site describes the product Manisyl which is the registered trademark of Sylka Managing Co. Inc.)
  22. www.vactup.org (Vaccine advocates. This site is a plethora of information about aids vaccines and other aids information)