Current State of Knowledge in Basic Sciences on HIV/AIDS

Luis Enrique Soto Ramírez

Introduction

The HIV/AIDS epidemic has had a great impact in the world not only because of the numerical magnitude of the problem, but because of a series of important collateral factors, such as the great impact on mortality of subjects in their productive and reproductive age, the great influence over human sexuality, and the disclosure of sexual behavior and preferences that were once kept in complete secrecy.

From the commercial point of view, all kinds of lucrative tendencies from pharmaceutical companies have emerged, and millions of dollars have been invested in the potential market that patients with HIV/AIDS represents. However, the results have been disappointing and, today, when drugs hold some promise, their production costs are so high that such drugs are out of the economic reach of most individuals who need them around the world.

From the scientific point of view, we have seen very important changes. A high number of researchers have changed their work areas, and, through the adaptation of their knowledge or the implementation of techniques already known, they have moved into the area of HIV/AIDS, where an important proportion of economic resources for basic research have been allocated. With these resources, a series of advances have been made on virology and molecular biology as never before in the history of science. What we know about HIV 13 years after its discovery is, without a doubt, more than what we know about any other infectious agent. Paralleling such scientific development, a vast laboratory industry has been created, with the marketing of equipment and chemicals for high-level research but at excessively high prices.

Fifteen years after the first case of AIDS was described in the United States, there is no cure nor a preventive vaccine for this disease. This has been a cause of frustration, desperation, and uncertainty. The consequence of these facts has been, despite the greater magnitude of the pandemic, the elimination of funds destined for research projects, which has led to the loss of interest from laboratories and pharmaceutical companies, especially on the design and testing of candidate vaccines. This has created a strong competition for these funds. In the middle of this crisis, even the once-considered great researchers on basic HIV science postulate ideas and concepts that are worthless a year later, casting doubt on the ethics of these researchers.

The competition, dubious ethics and commercialization put developing countries (including Latin America and the Caribbean) in a compromising position, especially at the attempt of developing basic research. The questions that must be asked are:

With the poor practical results obtained through basic research, must our countries invest in it or must we focus our resources on education, prevention, diagnosis, and the acquisition of new antiviral drugs?

In case of having the resources needed for basic research, which areas must receive higher priority?

To establish priorities, what kind of information must we rely upon?

In order to enhance our perspectives in this area of research, must collaboration with developed countries be established?

Who should take these decisions?

This document is a current review of important concepts related to basic research on HIV/AIDS, and it tries to give at least a partial answer to some of the questions already formulated.

Review of the state of knowledge the AIDS conference in Vancouver

Main advances on the subject

Four areas in basic science have been affected by most of the recent discoveries on HIV/AIDS: 1) advances on understanding the pathogenesis, especially at the level of receptors and cell tropism; 2) findings related to new therapeutic strategies; 3) the better understanding of genetic variability in HIV and consequently on molecular epidemiology of the virus, and 4) the perspectives on the development of vaccines.

Two receptors have been recently described, both actually involved as co-receptors of the CD4 molecule. The first is called fusin and is related to the entry of virus to lymphoid T-cell lines(1). The second is a receptor of the so-called beta-chemokines (cytokines which function as chemotactic factors of specific cell types), called CC-CKR5, which acts as a co-receptor for the entry of HIV into macrophages(2). This explains partially the inhibitory effect of beta-chemokines secreted by CD8 lymphocytes described recently(3). Therefore, the tropism for specific cell types is determined by factors on the host cell as well as by viral factors. The virus modifies its tropism with subtle or punctual changes in variable areas of the envelope protein (V1, V2 and V3), and probably through the multiple interaction among these regions(4). The differences in tropism by different viruses are reflected by many aspects of the pathogenesis of HIV but, more importantly, by their transmission. Thus, the target cells in the genital area (Langerhans cells) can determine the selection of certain variants or subtypes(5) and this can determine the predominance of such viral variants in a determined risk group.

Pathogenetically, the well known phenotype switch at the beginning of clinical manifestations, from non-syncitia forming to syncitia forming, could be explained by a switch of the preferential receptor from CC-CKR5 to fusin(6).

After many years of poor advances on therapeutics for HIV infection, it is now thought that HIV can be eradicated or at least inhibited for a long time. Three factors have contributed to these advances: 1) the better understanding of the pathogenesis: 2) the availability of quantitative methods to measure viral RNA present in plasma (viral load); and 3) the use of more powerful drugs.

Quantitation of viral load has been used to evaluate the equilibrium process between viral replication and availability of target cells. It has also been used for the characterization of the magnitude and duration of the antiviral effect of new drugs(7). Besides, viral load seems to be clearly related to progression of the disease(8,9).

Viral load can be determined by several methods. Among the first were the determination of p24 and quantitative cultures, but both of them were costly and inaccurate. Three methods are commonly used now; all of them are commercially available and use high-cost molecular technology. Reverse transcription of RNA (RT-PCR) in plasma can determine the quantity of viral RNA by comparing it with an internal standard. This technique has been commercialized by Roche laboratories (Amplicor HIV Monitor Test) and is probably the most sensitive of all, detecting up to 200 RNA copies per ml of plasma. The Nucleic Acid Sequence Based Amplification (NASBA) starts like the first technique, with a reverse transcription to generate multiple RNA molecules from the DNA obtained, which are then quantified. It can detect up to 400 copies of RNA per ml and it is commercialized by Organon-Technika (Q-NASBA). The third technique does not imply an amplification of nucleic acids and is named b-DNA for "branched DNA". In this case, RNA is fixed to a solid base and detected with DNA through lateral chains that allow a colorimetric detection. This low-sensitivity technique is commercialized by Chiron Corporation. With any of these techniques, the approximate cost is more than US $175 for each assay.

Combined regimes of nucleoside analogs lower viral load up to 10 times pretreatment values for up to 2 years, delaying but not preventing progression(10). The first results with the new protease inhibitors, when associated with nucleoside analogs, seem to reduce viral load more than 100 times for periods as long as one year in 90% of treated patients. The levels observed during treatment, which are well under detection capabilities of current techniques and well below the levels seen in non-progressors are encouraging(11). Little is known, however, about the development of resistance under those conditions(12).

The indication for the use of protease inhibitors and viral load monitoring is now clear, but the cost of both is so high that only 1% to 2% of patients that require them would use them in Latin America and the Caribbean.

The great genetic variation observed in HIV is clearly related to the pathogenicity of the virus, immune escape, and the development of resistance to antiretroviral treatment. However, its importance has been questioned by the discovery that antigenic diversity is inversely related to progression to AIDS(13,14). This finding is yet to be confirmed, since there is ample evidence that links diversity not only to pathogenicity and development of resistance, but to other phenomena such as differential transmission of some subtypes(5,15,16).

Genetic diversity of HIV is due to two very important events: mutation and recombination; both of them occur through a viral genome of 10,000 base pairs. Mutations are due to the inaccurate function of reverse transcriptase, an enzyme that replicates the genomic material of HIV. When this enzyme makes a mistake while inserting new nucleotides, it cannot go back to correct it. Approximately one mutation per viral cycle is produced, which at the end of 100 cycles produces diverse mutants. On the other hand, recombination is an apparently non-random event which involves the exchange of some regions between viruses from different subtypes. This originates very divergent variants in a single cycle. For recombination to occur between two different subtypes, they have to coexist in the same individual(17,18). The sequencing of specific regions of gag and env genes can be a good parameter to evaluate mutations, but it is not good enough to evaluate recombination, which requires the sequence of a complete genome and from an evaluation process called "bootscanning". Until now, only 32 complete viral genomes have been sequenced(19).

The genetic variability originated either from mutation or recombination in the regions of the viral envelope and the capsid (gag) is currently translated by the presence of at least 9 viral subtypes designated "A" through "I". These subtypes are classified into group M and are different from group O, a group of sequences very distant to any of the designated subtypes and in which classification has not been possible(19-21). Subtypes A through E currently have a characteristic geographic distribution. Subtypes A, C and D are predominant in Sub-Saharan Africa: subtype C predominates in India; and subtype E in Thailand. Subtype B is dominant in the United States and western Europe, in the Caribbean and in South America. This subtype predominates in geographic regions where homosexual intercourse or IV drug use are the main routes of transmission. In contrast, non-B subtypes predominate in regions where heterosexual intercourse is the main route of transmission(19,20). The biological significance of these subtypes has not been established. The single reference comes from the differential replication that exists in Langerhans cells between virus from subtypes B and E. Since Langerhans cells are the possible primary targets of heterosexual transmission in the genital area, the fact that they replicate subtype E better than subtype B suggests that this tropism could be the explanation to differential expansion of subtype B epidemics among IV drug users and E among heterosexuals in Thailand, as well as in other geographic regions(5) .

Heterosexual transmission is increasing throughout the world, and of course in Latin America and the Caribbean. Besides Brazil, where reports of subtype F and a non-official report from the World Health Organization in which the presence of subtype C in Honduras is revealed (specifically San Pedro Sula, where an outbreak of heterosexual transmission has been detected), little has been said about subtypes in this geographic region(22).

Finally, it is possible that other subtypes may arise in Western countries and may cause an epidemic of heterosexual transmission. This has been denied in the United States but has started to be proven in Europe.

On vaccines it is important to mention that due to the genetic and biological differences seen in different subtypes and the absence of serologic cross-reaction, it is accepted that the vaccines have to be subtype-directed, which accentuates even more the need to know the viral subtypes prevalent in different geographic regions(23).

A vaccine for HIV is the greatest hope to control the pandemic, especially because of the cost of current treatments and the uncertainty of their effectiveness in the long run. The basic questions for the development of any vaccine have not been answered for HIV. The immunologic response that must be created as well as the most adequate viral antigens to produce it are still unknown. Finally, an adequate vector to deliver the vaccine to the proper tissues has not been created. Today, more than 20 candidates are being evaluated in phase I clinical trials, and 2 or 3 are being evaluated in phase II; in both phases I and II only for safety and immunogenicity, but no candidates are being evaluated in phase 3 for effectiveness(24,25). Since the number of individuals needed to prove the effectiveness of a vaccine is directly related to the incidence of infections, effectiveness studies must be undertaken in developing countries with incidence rates from 6% to 16%(26). However, although several countries have been targeted for effectiveness studies (Brazil, Thailand, Uganda), there is no consensus on whether they must proceed, since candidate vaccines may provide unacceptably low protection.

Two lines of research have been recently introduced in the area of vaccines. Isolated viral DNA can be introduced in muscle tissue of animals, and an immune response to viral proteins can be elicited. Whether this response is protective or not is still unknown. No trials have been undertaken in humans and there is doubt on whether these integrated sequences might cause the emergence of malignancy(27). On the other hand, the use of live attenuated virus has cast some doubts. Although the general belief is that the use of attenuated viruses can be effective, there are not many studies on adequate attenuation forms, and if there were any, it is questionable if these viruses might revert to their original pathogenicity on superinfection, on recombination with retroviral sequences in the human genome, or through transference to neonates or immunocompromised hosts(28,29).

Main questions on investigation of the topic

The questions that arise from the advances presented above are:

1. Is antigenic diversity of HIV originated in the different receptors required for its entry into cells?
2. Which are the viral epitopes and cell receptors that interact?
3. Which is the role of beta-chemokines and other cytokines?
4. Are the current techniques for estimating viral load adequate for all viral variants?
5. How can these techniques be modified to make them cheaper?
6. Which new effective medications can be created at a lower cost?
7. Will treatment with protease inhibitors be able to eradicate the infection in sanctuary sites such as lymph nodes?
8. Which are the consequences of the great decrease in viral load of these drugs on viral variability and the development of resistance?
9. Are there any other viral subtypes in developed countries?
10. Which are the prevalent subtypes in Latin America and the Caribbean?
11. In case new subtypes are found, are they associated with heterosexual transmission?
12. Which are the viral determinants of higher heterosexual transmission?
13. Is tropism for Langerhans cells an applicable phenomenon to other subtypes or to subtype B variants?
14. Should we proceed with effectiveness studies (Phase III) with other vaccine candidates and where?
15. Which are the immunologic parameters that correlate with protection of an HIV vaccine?
16. Are DNA vaccines and live attenuated vaccines safe?

Main questions for the decision-taking process

These questions were partially sketched in the introduction. Of course, the most relevant for Latin America and the Caribbean is, Should basic research be undertaken? This question is related to cost and to the clear leadership that developed countries have in this area. If the answer to this question is affirmative, it is necessary to establish research priorities. This process must be undertaken by scientists who have current knowledge of recent advances and who have a broad point of view of the situation in the region. In any case, our goals in basic research will be limited, so should we establish collaboration with developed countries? If so, with which countries and under what conditions?

From all questions stated under section 2.2.2, I consider that the most important one for Latin America and the Caribbean is whether or not different viral subtypes should be studied in the area.

Review of material presented at the conference

For the purpose of this review, it was considered adequate to unify all suggested relevant points. Therefore, the main research themes presented at the conference are reviewed, highlighting those relevant for decision-making, as well as thematic advances of the conference and the best presentations (which are referred to with the original review number), and some negative examples that must be avoided.

New diagnostic, prognostic and follow-up methods.

a) Virologic. From the virologic point of view, several findings related to quantitation of viral load and proviral load were presented, as well as the extraction of complete genomes for their complete analysis, crystallization of protease and development of the molecule SHIV.

On viral load quantitation, the use of an RNA control is paramount. Since RNA is labile and can be easily degraded, the use of protected RNA to avoid degradation was presented and its reproducibility was evaluated(30).

In view of the dissemination of several viral subtypes (see section 2.5), it is very important that viral quantitation techniques are useful for all viral subtypes. The study L.B.A. 6005(31) was presented as evidence that the commercial kit that uses the "branched DNA" technique is able to detect and quantify all subtypes from A to F, while the kit that uses RT-PCR (Roche Diagnostic Systems) did not detect subtype A, and the values obtained with this technique were clearly lower for strains E and F than those obtained with the branched DNA technique(31). In the light of this information, it is important to note that the methods to be used or to be created in Latin America and the Caribbean must be directed to viral subtypes prevalent in the area.

The viral construct SHIV is a simian retrovirus into which the envelope (env) genes and the regulatory regions tat and rev from HIV-1 have been inserted. When inoculated in rectum, vagina and through the IV route in simians, it causes a rapid fall of CD4+ cell count. It is an excellent model for proving effectiveness of vaccine candidates(32).

b) Immunologic. From the immunologic point of view, the possible importance of chemokine receptors as well as factor CAF (Cell Antiviral Factor) was described. New vectors based on retroviruses and other viruses (Canarypox, adenovirus, etc.) as well as ex-vivo systems for T-cell CD3+/CD28+ expansion, and simple, fast and cheaper methods for the quantitation of lymphocyte subpopulations have been created.

c) Diagnostic. From the diagnostic point of view, new enzyme immunoassays were described, with special emphasis in the detection of group O. The dried blood spot technique was exalted, which can be useful to perform diagnostic tests as well as viral subtype determination assays(33).

In a new diagnostic approach, an agglutination technique which uses a peptide called TaBS as a target was presented. This peptide binds to the HIV-1 envelope (gp120) but not to HIV-2. The current results are reproducible in the detection of cell-cultured virus, but there are no data on its performance in serum or plasma(34).

Future reasearch trends in this section will have to include the development of lower-cost techniques for diagnosis and determination of viral load. On the other hand, new quantitative and reproducible assays must be created to evaluate cellular immune response, and the role of chemokine receptors in HIV infection and the general immune function must be studied.

Pathogenesis of HIV infection

For HIV to produce damage to the immune system and eventually AIDS, the participation of viral factors together with host factors is required. Even though viral factors seem to be more important, it is actually the interaction of both which gives the final result.

Viral factors

The constant viral replication is probably the most important force which leads to immune deficiency; proof of this is that viral load quantitation is currently the basic guide for prognosis of an infected individual. Highlighting the importance of viral factors is the fact that the size of the initial inoculum and the transmitted viral phenotype are directly related to the rate of progression to AIDS.

In viral replication and in the consequent death of CD4+ lymphocytes, some important factors were mentioned:

a) Entry and viral transport. Once the fusion of membranes (viral and cellular) has occurred, the viral nucleoprotein complex is recognized by the cell through cell components such as cyclophyline. This union induces the liberation of the viral genetic content(35). Later, once the reverse transcription has occurred, the proviral DNA is transported to the nucleus through the interaction of integrase and cell carriers called cariopherines(36). These findings open possible targets for antiretroviral therapy.

b) Replication. On viral replication, the most relevant news were the trials for its quantitation. By using HIV infection cases in specific hosts and through a better understanding of the dynamics of lymphoid cells, it has been calculated that an infected individual can produce up to 10 billion particles in one day. From 200 to 700 particles come from each infected cell(37). As a consequence, 180 new viral generations are originated per year. This is a clear quantitative explanation of the great viral variability, as well as of the easiness with which resistance to diverse antiretroviral treatments is developed. However, because these concepts may have a limited usefulness in the physiopathology of infection as well as in the development of therapeutic options, I consider them of low priority.

c) Infectiousness. The infectiousness of HIV has been related to the presence of the product of the regulator gene nef. This product interacts with a cell kinase activating the viral enzyme reverse transcriptase, which exerts a positive effect on viral infectiousness(38). On the other hand, it was demonstrated that the protein derived from gene nef is incorporated into the virions, where it is digested in two fragments. One of them, a 20 kD fragment protein, exerts a positive effect on HIV-1 infectivity. This mechanism could explain why no progression to AIDS is observed in individuals infected by nef-defective viruses, and why protease inhibitors have an infectivity inhibiting effect through blocking the digestion of nef in virions(39).

d) Viral dynamics. It was thought that the asymptomatic period of HIV infection was a period of viral inactivity. It is now clear that it consists of an equilibrium between constant viral replication and cell death with replacement of destroyed lymphoid cells. The forces that keep this equilibrium are, on one side, the immune response and the availability of target cells and, on the other, viral replication and viral genetic variability. In this context, CD4 levels represent a parameter of the distance between the actual state and progression to AIDS, while viral load represents the rate at which this change will occur(40).

Host factors

The importance of host factors is clearly highlighted in the relation between immune response and progression to disease which occurs during primary infection. In this respect, viral load does not correlate with progression, but the presence of a policlonal or monoclonal response in relation to the repertoire of receptors in T-cells does(41). Other factors considered relevant are mentioned below:

a) Receptors. Although the first receptor described for HIV is the so-called CD4, it seems clear now that other co-factors besides this receptor are required. The two CD4 co-receptors reported before the conference have been mentioned, fusin for T-cell tropic and syncitia forming viruses(36), and CC-CKR5 for macrophage-tropic variants(42). Peripheral lymphocytes probably have both co-receptors, while circulating monocytes and tissue macrophages only bear CC-CKR5. However, advances on the shape or exact function of these receptors and their interaction with HIV at a structural level have not been mentioned.

b) Types of infected cells. Besides the already known target cells, the infection of megakaryocytes(43) as well as kidney mesangial cells(44) has been described. These findings could explain the HIV-related thrombocytopenia as well as renal damage described in infected individuals. In mesangial cells, the infection activates the production of cytokines that can contribute to renal damage.

c) Transmission. In perinatal transmission the rupture of membranes has been described as a risk factor. Viral excretion in the genital area is also important. This excretion does not correlate with viral serum levels and could be due to a local production of virus originated in infected cells such as Langerhans cells in the mother since the time of infection or as a consequence of infection. This finding can be related to the predominance of non-syncitia forming phenotype in primary infection of children as a consequence of a preferential production of virus of this genotype in the genital area(45).

d) Immune function. Little is known about the real antiviral effect of the immune system. Many studies are focusing on the antiviral effects of cytokines, especially chemokines.

Interleukin 1 alpha and IL-6 as well as Tumor Necrosis Factor are cytokines produced abundantly in lymphoid tissue, which induce HIV replication. This positive effect is inhibited by IL-10 in vitro. Also, beta-chemokines such as RANTES and Macrophage Inflammatory Protein (MIP 1 alpha and 1 beta) directly diminish viral replication in some cell systems in vitro(46). This effect occurs at higher concentrations than those found in vivo. Even though it has been demonstrated that beta-chemokines have an antiviral effect in vitro, their association in vivo to the progression to disease or viral load has not been proven, so their role in the long-term control of viremia is doubtful(47). With these findings, it is hard to believe the efficiency of the proposal to use IL-10 immediately in patients to test its efficacy. This study, which has been practically accepted because it was postulated by one of the leaders in AIDS research, is one of the clearest negative examples in decision-making strategies.

Another factor observed to affect viral dynamics is CAF (Cell Antiviral Factor), produced by CD8+ cells and documented especially in non-progressors and non-infected high-risk individuals. This factor has not been identified as any of the known chemokines and it has a potent inhibitory effect on replication(48,49). In cases in which infection (replication) is controlled, there is an apparent equilibrium between CD4+ and CD8+ cells through the production of IL-2 by the former, which stimulates CD8+ cells, which produce CAF and finally inhibit viral replication(50).

The response of cytotoxic T-cells in primary infection showed a clear prognostic value on the course of infection. When this response is depressed, the prognosis is always grave, with rapid progression to AIDS.

HIV can also cause direct immune suppression. This effect is produced by multiple viral proteins, especially those in the envelope and even by proteins which originated in defective viruses, and it occurs through programmed cell death or apoptosis. The mentioned proteins stimulate non-infected peripheral blood mononuclear cells to produce a substance blocked by anti-lymphotoxin antibodies (also anti-fas or anti-IL-10). which originates a programmed cell death when the CD4+ cell contacts the antigen(51).

Treatment of HIV infection

The results presented at this conference speak clearly of the termination of monotherapy with Zidovudine (ZDV). On the other hand, the usefulness of initiating treatment at earlier stages (200-500 CD4+ cells per ml) especially if viral load is elevated, and the usefulness of the combination of nucleoside analogs (AZT/3TC, D4T/ddI and D4T/3TC) is confirmed. The major advance is probably represented by the results obtained with the new protease inhibitors. When used in combination (AZT/3TC/Indinavir), they markedly lower viral load, even to levels not seen before with other treatments (probable full inhibition) and for prolonged periods of time (48 weeks). These aspects, which are reviewed with greater emphasis in the clinical section, originate a basic question on the possibility of viral eradication and cure. The blockage of viral replication is now considered to be transitory, and may not reach cells outside of the circulation. Therefore, viral replication may reappear if treatment is terminated. In any way, if long-term inhibition is proven, this would have a greater impact on infected individuals. The search for answers to these questions as well as their impact in viral variability must create a special field of research.(40) .

Protection from perinatal transmission from mother to child with ZDV treatment, which had a great impact at the Yokohama conference, was confirmed, although advances on resistance and toxicity of this kind of treatment were very scarce(45) . No conclusion has been reached on dosage and ideal times of use in relation to effectiveness, cost, and collateral effects. The use of drug combinations and the inclusion of protease inhibitors, with the potential for higher replication blockage, could enhance protection and lower transmission to negligible levels.

Treatment with 3TC (Lamivudine), a nucleoside analog, increases the fidelity of reverse transcriptase(52). This effect could be related or augmented by the presence of characteristic mutations of resistance to 3TC, especially Met184Val(53). Therefore, besides partially blocking viral replication, unblocked partial replication generates viruses with less heterogeneity.

Safety and efficacy of new treatment combinations must be investigated in the future. New prospects, such as integrase inhibitors, must be developed as well as inhibitors of tat/rev. The real effect of reduction in viral load must be carefully evaluated, as well as the consequences in the evolution of the disease and the appearance of new mutations associated to resistance.

From the immunologic point of view, the study of treatments to restore immune function is under way. Two approaches seem to be highly promising: one involves the blockade of chemokine receptors, and the second implies the in vitro expansion of non-infected T-cells for later reincorporation into the infected patient along with replication inhibitors to prevent their infection.

Few results were presented on gene therapy, and they are not very promising, especially for ribozymes. On the other hand, two new approaches might hold promise. The first is the insertion of gene sequences that are inhibitory for viral replication into lymphoid mother cells which can later be allocated in infected patients(54). The second is the creation of nucleases which are active only against HIV RNA and can be transfected in human cells(55).

Resistance to antiretroviral treatment

Even though the most common cause of the development of resistance to antiretroviral treatment is the appearance of mutations, recombination is a phenomenon that must be considered, especially when several recombination points in the polymerase have been documented(56).

Mutations and recombinations exist at the beginning of treatment and they are selected throughout the duration of treatment. The concomitant events for the development of resistance have been useful to define concepts such as "viral fitness". It is clear that wild-type viruses are more capable of replication than mutants in the absence of the retroviral ZDV, but this relation is inverted in the presence of the drug (selection pressure, which increases with increasing doses of ZDV)(57). This clear difference in "viral fitness" depends also on the cell type. Thus, mutants of codon 84 have a lower replication rate in peripheral blood mononuclear cells (PBMC) than in T-cell lineages. Therefore, the use of PBMCs in all mutant viral dynamics assays is recommended(58). The concept of "viral fitness" also influences the continuing appearance of some mutations even after suspension of treatment(56), owing to the replicative advantages presented. Following this idea, there are clear differences in the development of resistance among medications which have limited suppression on viral replication (3TC), those with moderate suppression (protease inhibitors), and those in which suppression is complete or almost complete (double or triple combinations). In the latter case, if treatment was suspended, wild-type viruses could grow again but mutant types could not(40)^.

One of the objectives of treatment is to block resistance. This generally implies the use of at least two drugs, originating resistance to one of them and the permanence of non-resistant strains for the second. This is probably related to changes in the structure of reverse transcriptase as has been seen with nucleoside analogs, in which mutations of codons 215 and 219 are associated with resistance to ZDV (suppressor mutations). The combination of ZDV with ddI or ddC does not block the development of resistance to ZDV as it was originally thought. However, the combination of ZDV and 3TC in a patient without previous treatment originates mutation I84V which slowly confers resistance to 3TC and blocks the development of resistance to ZDV. In patients with previous ZDV treatment, resistance to 3TC develops rather quickly and susceptibility to ZDV is recovered.

Resistance to protease inhibitors is associated to multiple mutations, some of which could be specific or common for other drugs in the same group. In the case of Ritonavir and Indinavir, mutation V82A is fundamental for the subsequent development of other mutations, as mutation L90M is for Saquinavir. Sometimes, the presence of only one mutation could predispose to the development of rapid cross-resistance when changing from one protease inhibitor to another. In vitro studies have shown that for an apparent effect to occur on diminished susceptibility to Indinavir, at least 3 mutations are required. In any case, the effect of great viral suppression of combinations of nucleoside analogs and protease inhibitors has completely modified the idea of resistance, since it is very difficult under these conditions for viral variants with mutations associated to resistance to be replicated(59).

Vaccines against HIV

The most relevant advance in this area is related to the DNA vaccine. The doses (DNA quantities) needed have been lowered thanks to the use of cationic lipids (Vitamin D3) which favor DNA transfection in muscle cells(60). Safety and immune response have already been demonstrated in rodents, rabbits and primates, but possible oncogenicity is a matter of concern and cannot be forgotten since follow-up of these animals is short. On the other hand, protection data were presented against HIV in chimpanzees who were vaccinated with DNA from the envelope and from the gag/pol regions. In both cases, when these animals were challenged via the IV route, they remained free of infection according to RT-PCR for up to 26 weeks after the challenge, while controls were positive on the second week. Phase I trials in humans are under way(61) .

The work that demonstrated protection against HIV infection in rats with the use of a monoclonal antibody that neutralizes primary isolates from subtypes A through F (probably associated to a direct action of the antibody against an oligomer structure native of gp120), has to be taken with caution especially in view of the variable parameters available for the evaluation of neutralization and the low extrapolation from murine models to humans(62).

There still is a great vacuum on immunologic parameters that correlate with protection. Cytotoxic T-cells (CD8+,CD28-) seem to have little protective effect and they seem to be deleterious, even after being used as a parameter for response in vaccine candidates. In contrast, non-cytotoxic CD8 lymphocytes (CD28+) seem to be crucial.(50,63) On the immunity side, the latter’s importance is not recognized although the correlation between homologous neutralization and protection has been confirmed. New assays with direct neutralization of variable regions V1 and V2 from the envelope (not region V3) could help solve this deficiency, according to a finding presented on a marked variation of region V1 in relation to immune pressure(64).

Even though the importance of mucosal immunity has not been established, few advances have been made to stimulate it. Data on the development of mucosal antibodies after intranasal inoculation of a prime-boost scheme were presented, with adenovirus as a vector and a reinforcement with gp120 and intrarectal inoculation, but in both cases only an IgG response was observed(65,66). On the contrary, new DNA vaccines induce an adequate mucosal response mediated through IgA(60).

Many aspects were left for the future. It is clear that all these vaccines must be directed against more than one viral subtype, which was demonstrated by presentation(67) in which protection of vaccines with viral subunits was only subtype-specific. Immunization with two viral subtypes with protection against both was found also. In addition, the best immune response has been obtained with the Priming and Boosting technique, that is, an initial dose of a vaccine with a vector reinforced by subsequent doses of a single viral subunit(68).

New quantitative immunological evaluation tests are necessary, especially on the cellular branch to evaluate the effect of vaccine candidates.

Research on effectiveness of live inactivated vaccines in animals was presented(69), but there was no mention of the possible reversion to pathogenic strains, or new models of viral inactivation.

With all these candidates, new phase I and II trials in humans must be created. Interestingly, phase II trials seem not to be associated to a higher frequency of risk behavior. However, the cases of concomitant infection in these trials have not been completely studied, since these studies were not designed to evaluate effectiveness(70). Whether vaccines with no favorable immunologic parameters must proceed to phase III effectiveness trials is not clear up to this point. This was a subject of great debate: concerning developing countries with a high HIV infection incidence, a vaccine which provides low protection would have a great effect on cost and life expectancy (71). This argument is valid if we consider that there are no advances on protection parameters, for which a phase III effectiveness trials would provide a positive or negative answer. On the other hand, the resources for these trials have to and will originate in developing countries, which are not in a hurry to start them(72). With any option, the consensus is that we must move faster to avoid more consequences of the pandemic.

Genotypes or international HIV subtypes

Genetic diversity of HIV is given by mutation and recombination. Evidence on selection during recombination was presented. Some sequences are observed to be preferentially retained, as is gp41 from subtype A which is also present in subtypes E, G and in A/D recombinants, suggesting some selection for that segment(73). It is important to consider that for recombination to occur, different subtypes must infect the same individual. This was considered to be uncommon, but it may not be so, especially in geographic regions where multiple subtypes are found. Thus, in Cameroon, double infection with HIV-1 and HIV-2 has been documented, as well as infection with different HIV-1 subtypes including group O, and one case of triple infection, the first reported(74).

According to 32 totally sequenced viral genomes, the real subtypes have been determined to be A, B, C and D, while E and G are recombinants. There is not enough information available for subtypes F, H, I and J. Ten percent of the sequences reported up to this day are recombinant, but these recombinants may predominate in the future. Clear recombination avenues have been established, and shown in the figure below:

The original distribution, which described well localized and geographically limited subtypes, is less valid now. Besides the possibility that in the future epidemics may be not only subtype specific but also recombinant specific, a diversification and dissemination of subtypes has been shown in this conference, especially a considerable expansion of non-B subtypes. Subtypes A, C, D, F and G were described in Europe; subtypes F and C in Brazil and Argentina;(75) and subtypes F and D in Mexico(76). In the Bronx (NY), 20% of subtypes were non-B; 5 were subtype A; one was subtype C; one B’ and one was non-typable (77). The first group O infection in the United States was described. Although this patient probably acquired the infection in Western Africa, it is relevant since it can be an index case for other individuals in the United States(78). Infections with group O were reported in 7 African countries which did not have this infection before. This is very relevant due to the difficult diagnosis for group O infections(79) and suggests a need to include specific peptides for this group in diagnostic equipment.

In general, subtype B is still predominant in Latin America. However, there are some particular characteristics of these viruses, such as the sequence GWGR in the crown of region V3 in Cuba(80) and in Brazil(81). No typifications performed in Honduras were presented, although the results obtained in this country are very similar to Mexico with a predominance of subtype B, and a few subtype D viruses which have important homology with subtype B (Dr. Myron Essex, personal communication).

Besides the great importance that this has for the development of adequate vaccines, viral variation has been correlated with biological differences. In this conference, two presentations dealt with the relationship between viral subtypes and certain phenotypes. Langerhans cells, which are one of the primary targets in the genital area in heterosexual transmission, replicate subtype E (published previously) and subtype C better than subtype B. Parallel to this, evidence that showed that the receptor for the virus on these cells is not CD4 was presented. This characteristic tropism could be the explanation to epidemic differences which occur in regions where heterosexual transmission occurs(82). Evidence that supports this finding is that in intravaginal inoculation of HIV in chimpanzees, with similar infecting doses with a mix of subtypes E and B, 6 weeks later all analyzed viruses were subtype E(83). This phenomenon is reflected on what has occurred in Thailand, where in 1991, 86% of sexually transmitted cases were due to subtype E and 14% to subtype B. For 1994-1995, the figures had changed to 98% E and 0.7% B. This has also been reflected in changes among IV drug users, in which subtype E represented 24% in 1991 and 44% in 1994-1995, as a consequence of the great predominance of subtype E in the population(84,85)^, .

Besides, the greater transmissibility of non-B viruses makes us think that other subtypes could cause a second epidemic in heterosexual population, as it occurred in Thailand. In Belgium, viruses A, C, D, F and H have been reported to predominate in the heterosexual population, although in low numbers, probably related to a recent introduction(86). A similar phenomenon is occurring in France (F. Barin, personal communication).

The importance of variants associated to a route of transmission was established through identifying infected children with drug-addict mothers, epidemiologically unrelated and with very similar sequences, which supports the presence of a social network indirectly associated through IV drug use(87) .

Even though presentation Tu.A2066(88) shows a higher pathogenicity for subtype E than for subtype C, there are some differences in this study that do not support their conclusions, such as the phenotype, and, in general, the evidence supports that none of the subtypes is more pathogenic, but they have adapted to certain routes of transmission.

Also, the presence of in vivo recombination is important, since viruses with fragments of two viral subtypes have been reported. Geographic borders do not seem to exist, but a predominance of subtypes or recombinant variants through regions or risk groups does, which will be very relevant on the design and application of vaccines.

The appearance of new variants in the future is expected. This has started to be seen in areas with great dynamics of transmission such as India, where the predominant subtype C has now two variants, C2 and C3. The first one is probably older due to the genetic variability it presents, and the second one may probably be a recently appearing variant(89). This can explain the different results published on longevity of the Indian epidemic. In Thailand, subtype B is actually a different variant denominated B’ to differentiate it from the traditional group B from the United States and Europe(84). This subtype B’ has disseminated from Thailand to the southern part of China, which has also received subtype C strains predominantly through IV drug users. Burma has received subtypes B’ and E from Thailand(90) .

From the technical point of view, it is important to mention that although good results on methods such as serotypification, heteroduplex mobility assay, and DNA hybridization have been published(91), the gold standard for viral typification is sequencing, especially from regions over 1500 base pairs. This will be even more relevant as subtypes start to mix and the detection of specific sequences related to certain pathogenic characteristics becomes more important.

Unanswered questions in areas under research

Problems not mentioned in the literature or during the conference

The information generated until this conference has importantly highlighted the presence and distribution of several HIV subtypes. However, some consequences of this information have been poorly referred to or not at all. Even though the detection of group O has been included in diagnostic kits, little has been said about the effectiveness of those kits with other subtypes and their consequences on quantitation of viral load have just started to be evaluated. Little emphasis has been placed on the presence of double infections with two different HIV-1 subtypes, and only two presentations have mentioned the biological importance of such subtypes.

Finally, although plenty of information on viral subtypes in different regions of the world is available, very few information is available from Latin America and the Caribbean, and especially from Honduras, where the existence of subtypes common in Asia is suspected. Although the processing of information from Honduras is underway (obviously originating in American institutions), the lack of interest from the United States towards what endemically occurs in Latin America and the Caribbean is remarkable, although migration and the description of non-B subtypes in these areas could make their way to this country.

It is surprising that two aspects particularly important at the Yokohama conference have not generated more information, and which I consider as still important. The first one is on mother-to-child transmission, which is a growing problem throughout the world. Little advance has been done on the mechanisms involved as well as the adverse effects that treatment with ZDV could carry. The second is the apparent standby of information generated by non-progressors, and for those who have been exposed several times but have not been infected. Although the answer to the immune parameters against infection or against disease lies in them, no advances have been done about this.

The absence of immunological parameters of protection has importantly delayed the development of vaccines. Even with new attractive candidates, the ambiguity from decision makers to initiate effectiveness trials is remarkable. On the other hand the information generated from phase I and phase II studies has not been completely used, noting the absence of information about concomitant infections which have appeared in such studies. Finally, and owing to the great importance that mucosal immunity might have, the lack of interest from some vaccine designers on this field is remarkable.

Useful conclusions for decision makers

The existing vacuum on HIV/AIDS basic research must orient decision makers. For those in developed countries, where most of this research is done, it will be important to establish support policies for research projects that have as an objective the covering of forgotten priority areas . These areas can be determined through expert panels which must be organized in a periodic fashion. For decision makers in Latin America and the Caribbean, these examples must help establish a coordinated basic research which is non repetitive, oriented to priority aspects as those mentioned in the conclusions.

Conclusions and Recommendations

Basic science applied to HIV is one of the least explored fields in Latin America and the Caribbean, which is directly related to the lack of resources allocated to this goal and the high costs involved. As clear evidence of this, from all presentations at the XI International conference, only 11 originated in this geographic region under label A of basic research. Interestingly, all of them referred to viral typification carried over in Mexico, Argentina, Brazil and Cuba.

Due to this limitation, it is probably difficult to make decisions on policies and actions to carry out, because, besides being very selective, they must be right.

The most important advance until this conference is the combined treatment with protease inhibitors. This treatment¾ excessively expensive according to pharmaceutical companies because of their high production costs¾ is practically out of reach for most individuals who need it due to the advanced stage of their disease. Little can be done to control the prices of these drugs, but even that must be done. It will be wise to lower taxes related to these products and obtain better prices for health institutions that provide them. However, a greater coverage is not probable since total cost is around 80,000 Mexican pesos per patient per year, and the need for treatment is on a long-term basis.

The future becomes darker when we take into account that to evaluate this and any other kind of therapeutics a costly assay such as viral load is needed. This assay allows not only to evaluate treatment, but to establish a prognosis on progression to disease; so. it is a high priority to make it available. Again, policies that favor the import of required equipment for quantitation of viral load and its sale at low prices must be considered. In this case, however, we have another weapon. Research for the development of cheaper alternate or similar techniques must be supported. Although these recommendations could apply for the quantitation of CD4+ lymphocytes, which is now clinically used for prognosis, besides the existence of candidates for cheaper techniques, it is clear that the prognostic superiority of viral load might be supported with a high priority.

With this point of view invaded by high costs, it is clear that the best option for control and prevention of HIV/AIDS is at the present time education, and for the future, the possibility of a preventive vaccine at low cost and that can be applied massively. It is important to point out that education for prevention must be maintained, since it will still lie at the center of control of the epidemic even with a vaccine and especially if this vaccine was not 100% protective, as it might happen.

At the present time, we must continue educating, but it is necessary to take other decisions that will be important for the future. The first of them is the participation of Latin American and Caribbean countries in phase III effectiveness trials for vaccine candidates. I believe it is clear that we must participate in them as long as there is a solid foundation for their use. On the other hand, it must be clear that according to the number of new cases occurring in this region, with one exception, none of our countries has the adequate scenario for these trials. The most important reason is that the fewer the number of cases, the greater the number of subjects who must be enrolled and the longer the time to obtain an answer about effectiveness of the tested candidate. Thus, African or Asian countries, such as India or Thailand, seem to be more adequate for these trials, and maybe the latter might start a phase III trial this year or early the next year. The exception is Brazil, which has been used as a target by the WHO for effectiveness studies and in which an adequate infrastructure has been created, although the information that supports this fact is scarce.

On the other hand, according to the information on genetic variability of the virus and the presence of several subtypes in different geographic regions without a specific pattern, each country or at least a region must be prepared to face the creation of an effective vaccine. If this vaccine is, as it has been mentioned, subtype-directed, it is a priority to establish in Latin America and the Caribbean adequate facilities for the optimal typification of HIV isolates. When I speak about optimal, I believe it is important to make some technical considerations. There are many ways to typify HIV, but according to the objectives through which we establish priorities, it must be done through nucleotide sequencing of the viral envelope of selected isolates to have a complete information on their genetic variation, especially on the region related to virus entry into its target cells. It is clear that variation is higher, and that specific strains are found in some countries or regions, such as B’ in Thailand. We have commented on the fact that subtype D viruses similar to subtype B have been found in Mexico and Honduras, but at the present time they have not been described in other regions. Besides, we must not forget that when two or more subtypes are found within a region, recombination can occur, and this gives rise to the emergence of new variants. Profound knowledge of the genetic information of our isolates will allow for the establishment of an adequate response to a vaccine that could be effective as created, or through modifications according to particular characteristics of each region or country.

This typification must be done in an oriented way. We have seen that certain subtypes have preference for specific transmission routes, and this was confirmed in one of the Mexican presentations, so a careful epidemiological study of risk groups must accompany a selection of representative cases from the population for typification since, technically and economically. this is a technique that cannot be applied massively. This fact is clear in Honduras where an epidemic of heterosexual transmission has been reported and where the University of Miami has established an outstanding epidemiological study from which some samples have been taken for typification at Harvard University.

More sophisticated studies as tropism or affinity of some viral variants for target cells, such as Langerhans in the female reproductive tract, must become high priority, since they will help determine viral pathogenic factors, and factors from the host particular to our region.

I believe that we must consider as high priority to include remote regions in our countries in the system searching for the adequate control of blood and blood derivatives to be used clinically. There is a considerable number of HIV infection cases originated in the deficiency of a test or an inadequate realization of the test. A system must be created which avoids this possibility, with an adequate distribution of equipment, training of personnel, and supervision. Besides, the use of equipment with adequate quality control and which fulfills all possibilities of viral subtypes present in the world must be emphasized. It is possible to create incentives for the development of equipment and chemicals at lower costs that may not jeopardize sensitivity or specificity, and whose quality is adequately controlled.

Finally, the concrete responses to the questions of our introduction are: Yes, we must invest in basic research on HIV/AIDS in Latin America and the Caribbean, but with clear objectives. Among the areas considered of high priority are the possible development of cheaper techniques for the establishment of prognosis and follow-up of treatment of patients with HIV/AIDS and for the adequate diagnosis of carriers of such infection and of contaminated blood products, the adequate typification of viral variants in the region, and the study of pathogenic factors of those variants. To do this, it is necessary for decision makers to directly rely on experts in this basic field, who must have up-dated information, as well as to create collaboration with prestigious foreign institutions, always under equal terms. It is even more important to strengthen the communication among groups who undertake basic science research in each country and in each region to avoid double functions, a common but sad fact due to the scarcity of resources.

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79. Saragosti S, Loussert-Ajaka I, Mauclere P, Descamps D, Bouchaud O, Simon F, Brun-Vezinet F. Genotypic and phenotypic charracterization of HIV-1 group O strains. XI International Conference on AIDS; 1996 julio 7-12; Vancouver, Canadá. (Abstract TuA.371).

80. Gomez CE, Iglesias E, Fernandez J, Lobaina L, Noa E, Diaz H, et al. DNA sequence of the C2-V3 region of the external glycoprotein (gp120) from Cuban HIV-1 infected individuals. XI International Conference on AIDS; 1996 julio 7-12; Vancouver, Canadá. (Abstract TuA.2057).

81. Casseb J, Hong M Gonsalez C, Duarte A, Hendry RM. HIV-1 serotyping among a cohort of individuals from Sao Paulo city, Brazil. XI International Conference on AIDS; 1996 julio 7-12; Vancouver, Canadá. (Abstract TuA.2082).

82. Soto-Ramirez LE, Renjifo B, Marlink R, McLane MF, Essex M. Differential growth of HIV-1 in Langerhans’ cells. Relation to transmission route. XI International Conference on AIDS; 1996 julio 7-12; Vancouver, Canadá. (Abstract TuA.370).

83. Girard M, Barré-Sinoussi F, Tartaglia J, Van der Ryst E, Paoletti E, Nara P., et al. Immunization with an HIV-1 Canarypox virus recombinant confers protection against genital HIV-1 challenge in chimpanzees. XI International Conference on AIDS; 1996 julio 7-12; Vancouver, Canadá. (Abstract We.A.393).

84. Subbarao S, Limpakarnjanarat K, Bhumisawasdi J, Young NL, Kalish ML, Schochetman S, Mastro TD. Genetic diversity od HIV-1 in Thailand 1994-1995. XI International Conference on AIDS; 1996 julio 7-12; Vancouver, Canadá. (Abstract TuA.375).

85. Mastro T, Ungchusak K, Vanichseni S, Young N, Limpakarnjanarat K, Raktham S, et al. The evolution of HIV-1 subtypes B and E in heterosexuals and injecting drug users (IDUS) in Thailand 1992-1995. XI International Conference on AIDS; 1996 julio 7-12; Vancouver, Canadá. (Abstract We.C.342).

86. Fransen K, Buv A, Nkengasong J, Janssens W, Heyndrickx L, Colebunders R, et al. The distribution of HIV-1 subtypes in the Belgian population. Europe is different from the United States of America. XI International Conference on AIDS; 1996 julio 7-12; Vancouver, Canadá. (Abstract We.C.343).

87. Kalish ML, Wiener P, Nesheim S, Lee F, Meadows L, Grimes V, et al. Detection of phylogenetically linked HIV strains among a population of epidemiologically unrelated women. XI International Conference on AIDS; 1996 julio 7-12; Vancouver, Canadá. (Abstract TuA.2078).

88. Shioda T, Oka S, Xin X, Liu H, Harukuni R, Fukushima M, et al. V2 extension of HIV-1 associated with slow/moderate disease progression. XI International Conference on AIDS; 1996 julio 7-12; Vancouver, Canadá. (Abstract TuA.2066).

89. Gadkari DA, Moore D, Sheppard H, Mehendale S, Kulkarni S, Bollinger R. Viral subtype analysis of HIV-1 infected patients from Pune, India. XI International Conference on AIDS; 1996 julio 7-12; Vancouver, Canadá. (Abstract TuA.374).

90. Shao Y, Zhao Q, Guan Y, Zeng Y, Chang J, Koestler J. Wolf H. Follow-up studies on molecular epidemiology of HIV-1 strains in Ruili region of southwest China. XI International Conference on AIDS; 1996 julio 7-12; Vancouver, Canadá. (Abstract We.C.341).

91. Luo CC, Downing R, De la Torre N, Candal D, Hu D, Otten R.A., et al. The evaluation of probe hybridizarion method used for the large scale screen of the distribution of HIV in Uganda. XI International Conference on AIDS; 1996 julio 7-12; Vancouver, Canadá. (Abstract We.C.344).