Abstract - When its T-lymphocyte host cell is activated, the latent (DNA) form of human immunodeficiency virus (HIV) is activated to produce RNA copies which are liberated as virus particles from the cell. In this process the cell is destroyed together with the latent virus. If administered at this time, 3'-azidothymidine (AZT) would specifically prevent the liberated RNA copies replicating and establishing latency in new host cells. The RNA copies would then be degraded by viral or host ribonucleases. Thus, one DNA copy of HIV and its RNA progeny would be eliminated from the body.

       Unlike most renewable end cells in the body, the maturation of T-cells involves processes of positive and negative selection. To preserve the 'educated' T-cell population, T-cell renewal occurs at the end cell level, rather than at the stem cell level. It is possible that normal physiological signals concerned with this homeostatic regulation of T-lymphocyte population size could be harnessed to produce synchronous activation of all T-lymphocytes. Tumor necrosis factor-alpha (TNF) has some of the properties expected of a postulated polyclonal activator needed for this programmed activation of T-lymphocytes.

Prolonged treatment with 3'-azidothymidine (AZT) extends the life of patients with AIDS who are infected with the human immunodeficiency virus (1). Recent evidence suggests that asymptomatic HIV-seropositive individuals can also benefit from prolonged AZT treatment (2). The continuing exponential increase in the number of seropositive individuals and the need for prolonged treatment with this expensive drug threatens to seriously burden healthcare systems worldwide.

      To be sure of curing AIDS, all forms of HIV within the body, both free and latent, need to be eradicated. Although there are many recent reviews on AIDS treatment strategies (2 - 6), to our knowledge none of these considers the possibilities that there might be conditions under which AZT could be employed:

• (i)   For short periods.
  
  

• (ii) To eliminate HIV entirely from the T-lymphocyte population.

We here consider various aspects of lymphocyte biology, the HIV life-cycle, and the mechanism of action of AZT, which lead us to a more optimistic assessment of the role of AZT and related drugs in the treatment of AIDS.

The 'education' of T-lymphocytes involves both positive and negative selection (7, 8). Positive selection generates sets of T-lymphocytes with the potential to respond to various 'self' determinants (e.g. MHC, CDI, TI and Qa-1 antigens; 9-11). Negative selection eliminates cells responding to self with high specificity. The final immunological repertoire consists of numerous small clones of cells. Members of a particular clone are each capable of recognizing a particular set of 'nearself' antigenic determinants with varying degrees of specificity.

      The range of specific responsiveness exhibited by an individual reflects the outcome of these selection processes (and further positive selections by foreign antigens), over many years. To renew the educated T-lymphocyte population after depletion (perhaps due to haemorrhage), could be a protracted process if renewal required reeducation. Individual T-cells (end cells), rather than stem cells, should be responsive to homeostatic control mechanisms affecting the size of the total T-lymphocyte population (7). Thus peripheral immunologically-competent clones of T-cells should be responsive not only to the cues provided by foreign antigenic determinants (through the determinant-specific T-cell antigen receptor) but also to cues provided by the growth factors concerned with T-lymphocyte population size homeostasis (through appropriate receptors). This self-renewing property of the peripheral T-lymphocyte population is now established experimentally (12-14).

Like other retroviruses, HIV integrates into the DNA of its host-cell and can remain there in quiescent form for prolonged periods (15, 16). Activation of latent viruses generally requires concomitant activation of their host cells, which then become permissive for virus production (17). In the case of HIV-infected 'resting' T-lymphocytes this activation appears to require reaction with antigen or lectin (18). Subsequent intracellular signals result in rapid changes in at least one protein encoded by a host gene (NFkB; 19). This protein may then transmit activation signals to other host genes which play a role in the switch from the resting (G₀) phase to the activated (G₁) phase of the cell cycle and/or in progression through the G₁ phase (20, 21). The protein may also transactivate HIV genes (22, 23). The activated HIV genome (DNA) is then transcribed to generate RNA copies of itself which are eventually packaged and released. The host cell with its associated HIV DNA is destroyed in this process.

     Thus cell activation is therapeutic to the extent that the latent DNA form of the virus is destroyed. The virus is triggered to destroy itself. The problem, of course, is that newly liberated viruses in RNA form infect new cells and can then establish latency in these cells. Effective therapy of AIDS requires a drug, or drugs, which can achieve two, preferably concomitant, results:

• The first of these is activation of all host cells carrying latent virus so that such cells will be destroyed by the virus.
  
  
• The second is the prevention of liberated viruses infecting, replicating in, and establishing latency in, previously uninfected cells

The ideal therapeutic agent is a 'magic bullet' which exploits some difference between the metabolisms of a pathogen and its host. The replication of HIV is strictly dependent on the viral enzyme reverse transcriptase, which is a DNA polymerase generating DNA from the viral RNA template. This enzyme is widely distributed in both eukaryotes and prokaryotes (24), but has not been shown to play a critical role in eukaryotes. The enzyme differs from the major host DNA polymerase activity in not being in a multienzyme complex (25, 26), and not being capable of proof-reading DNA. The nucleotides which are linked together linearly to make DNA are not scrutinized for abnormalities (27, 28). The resulting high error rate results in a diversity of forms which may be advantageous for the virus (29).

       The most widely accepted view of the selectivity of the action of the nucleotide analogue AZT is that it is added by the viral reverse transcriptase to the elongating copy of viral DNA, but is rejected by the proof-reading activity of host DNA polymerase. Thus replication of HIV-RNA is selectively interrupted (3, 4, 30). Ribonuclease associated with the reverse transcriptase, or a host ribonuclease, would probably then destroy the RNA so that it could not act as a template for more copies of itself.

The switch of a G₀ T-lymphocyte containing HIV DNA in its genome to the G₁ state permissive for viral replication can occur under two circumstances:

• The cell can be activated by an antigenic determinant corresponding to the cell's specific antigen-receptor (monoclonal response). The timing of this event would be entirely dependent on random chance encounter of the infected person with that particular antigen.
  
  

• Alternatively, the cell can be activated as part of a polyclonal homeostatic response to a lectin-like factor (lymphokine, growth factor) which signals a need to restore the size of the T-lymphocyte population (or a subpopulation). The timing of this event could be orchestrated by, for example, administration of the appropriate factor.

  Currently, treatment with AZT must be prolonged because the triggering of latent HIV (DNA) to destroy itself is mainly dependent on G₀/G₁ switches generated by random antigenic signals. At one point in time only one cell (or a small group of cells), becomes permissive for HIV replication. The cell, with its integrated HIV DNA, is then destroyed. AZT prevents the liberated viruses replicating and establishing latency in fresh host cells. However, if all host T-lymphocyte were activated synchronously by an appropriate concentration of an appropriate growth factor, all the integrated HIV DNA molecules would be destroyed with their host cells. Furthermore, all liberated RNA viruses could be prevented from replicating in previously uninfected cells by a short and intensive concomitant course of AZT. For this purpose, the effectiveness of AZT might be increased by combining AZT with drugs (e.g. 5-fluorodeoxyuridine; 25, 31), which deplete intracellular pools of natural AZT competitors and reduce feedback inhibition of the enzyme which is rate-limiting for incorporation of AZT (32).

     While much remains to be learned about the normal signals affecting cell population size homeostasis, there are indications that this 'one shot' approach to AZT therapy is feasible. Matsuyama et al (33) have suggested that a suitable polyclonal 'growth factor' might be tumor necrosis factor-alpha (TNF-alpha). This can activate host cell NFkB-like factors which, in turn, can activate latent HIV (34, 35). Treatment with TNF-alpha alone would be expected to accelerate the progression of AIDS (36). Concomitant treatment with AZT might turn this progression to the advantage of the host.

The above discussion is concerned with eliminating the AIDS virus from the T-lymphocyte population and thus preventing or correcting acquired immune deficiency. The virus can also persist in various other tissues, thus constituting a reservoir from which reinfection of T-lymphocytes might occur (16). Cotherapy with AZT and an appropriate end-cell specific cytokine or growth factor, to convert the cells to a state permissive for viral growth, might eliminate this reservoir (37, 38).

• "The presence of this latent reservoir of HIV is of considerable concern since these cells remain as a potential source of reactivation of viral replication."

  
  

• "Since it is likely that ... infected cells die upon activation of virus and that HAART prevents spread of virus to adjacent cells, the observation that this combination of cytokines [IL6, TNF-alpha, IL2] can markedly induce viral replication in this reservoir may have important implications for the activation-mediated diminution of the latent reservoir of HIV in patients receiving HAART."

  
  

• "Since cytokines alone can reactive HIV-1 replication in latently infected, resting, CD4+ cells, and since these cells probably die upon reactivation, it is conceivable that a strategy of administration of cytokines together with HAART might result in a diminution of this reservoir of latently infected cells".

  
  

• "Given the relatively long half-life of these latently infected, resting CD4+ T cells, and the fact that they are constantly within an environment capable of providing the stimuli for reactivation, it is not unreasonable to explore strategies aimed at deliberately diminishing the size of this pool of cells. In this regard, since cytokine-mediated induction of HIV-1 replication in these cells with subsequent release of virus probably results in death of the cell, and since the presence of HAART in vitro prevents spread of released virus, it is conceivable that in vivo administration of cytokines together with HAART may have such an effect."

• "Infectious HIV-1 persists latently in resting, memory CD4 lymphocytes in a post-integrated form despite 1 to 2 years of combination therapy [i.e. AZT or similar compounds + protease inhibitors]. This latent reservoir of HIV-1, denoted "L" , ... represents the major documented hurdle to virus eradication, although other ... viral sanctuaries may exist."

  
  

• "5 to 7 years of continuous, completely inhibitory therapy will be necessary to eliminate L. Treatment interruptions that permit HIV-1 replication to resume will rapidly restore the size of L. For larger pool sizes ... more than 10 years of continuous treatment will be required. A treatment duration this protracted is unacceptable because of the complexity, toxicity and cost of the current drug regimes... ."
   

• "Infectious proviruses are undoubtedly harbored within a diverse population of resting CD4 lymphocytes with memory for a large array of exogenous antigens. Thus, administration of a limited set of antigens is unlikely to activate a sufficient number of these cells to replicate and thereby facilitate their rapid death. On the other hand, the use of a large panel of antigens would be impractical.
  
          What about the administration of cytokines? Interleukin-2 (IL-2) alone is not expected to activate resting [G0] lymphocytes because such cells do not express the appropriate high affinity receptor. However, mixtures of certain cytokines, such as IL-2, IL-6 and tumor necrosis factor, have been shown to activate resting T cells in vitro. "

COMMENTARY (with copyright permission from the publisher Alexander Grimwade)

HIV: A Grouse-shooting Analogy

By Donald R. Forsdyke

The Hot Papers article¹ of Dec. 6 on the failure of various combinations of antibiotics to eradicate latent HIV gives the false impression that AIDS researchers were not aware of this possibility. ("Scientists are still grappling with the questions raised by this sobering discovery.")

    Doctors learn at medical school the fundamental rule that antibiotics should be given for short periods in adequate doses to destroy all pathogens and prevent the emergence of resistant strains. As soon as it was appreciated that AIDS was caused by a retrovirus, it was predictable that antibiotics alone would be unlikely to work. Retroviruses usually have a latency option and are highly prone to mutate.

     Thus, future therapy would have to be rather like grouse-shooting; one would need guns to shoot the birds and beaters to flush them out. To rid an area of grouse neither alone suffices. The combination is lethal.²

     Accordingly, the research agenda for AIDS had to develop along two lines:

• (1) research on viral growth and replication to find antibiotics such as AZT, which would hit the virus "on the wing," and
  
  
• (2) research on latency to find drugs such as TNF-alpha, which would flush out viral reservoirs.

A short and inexpensive period of treatment should then suffice (expense being a particularly important factor in Third World countries).

     Unfortunately, our research systems do not operate this way.^3,4 Antibiotics were emphasized rather than latency-disrupting drugs. When AZT did not come up to expectations and resistant strains emerged, the call came for bigger and better guns, rather than for beaters. If regular guns won't work, add the howitzers! If that combination does not work, add the mortars! And to forestall criticism, call it "highly active anti- retroviral therapy"  (HAART)!

     The quite predictable consequence is that we have a variety of initially highly effective antibiotics to which HIV is now resistant. Thankfully, HAART can increase the life span of patients, but these same patients, by virtue of the resistant strains they harbor, remain as potentially dangerous sources of infection. When latency-disrupting drugs eventually emerge (and let us hope the work on IL-2 is not just hype), it may be too late. HIV may be totally resistant.^5,6

     Part of the problem is a refusal to use the word "antibiotic" in the context of HIV treatment. Instead, patients are subject to antiretroviral therapy with "agents" or "drugs." Furthermore, it is argued that "therapy for HIV-1 disease can be viewed in a way that is similar to treatment for cancer," instead of similar to treatment of other pathogen-caused diseases.⁷

References

1. S. Bunk with comments by D.D. Richman and R.F. Siliciano, Hot Papers, The Scientist, 13[24]:22, Dec. 6, 1999.

2. D.R. Forsdyke, "Programmed activation of T lymphocytes: a theoretical basis for short term treatment of AIDS with Azidothymidine," Medical Hypotheses, 34: 24­7, 1991.

3. D.R. Forsdyke, "A systems analyst asks about AIDS research funding," Lancet, 2:1382­4, 1989.

4. D.R. Forsdyke, "Bicameral grant review: how a systems analyst with AIDS would reform research funding," Accountability in Research, 2:237­41, 1993.

5. D.R. Forsdyke, post.queensu.ca/~forsdyke/aids.htm

6. D.R. Forsdyke, Tomorrow's Cures Today? Newark, Harwood Academic, 2000. (Click Here)

7. R.J. Pomerantz, "Residual HIV-1 disease in the era of highly active antiretroviral therapy." New England Journal of Medicine, 340:1672­4, 1999.

[Prefers to remain anonymous]
Professor
[                      ]
Department of Genetics
University of [   ]"
USA

A similar point was made in The Scientist March 20th (Click Here), which was briefly replied to in the April issue: (Click Here)

Definition of "Antibiotic"

Thank you for your comment concerning "the standard term". Webster's Dictionary defines "antibiotic", when used as an adjective, as something "tending to prevent, inhibit, or destroy life." As you imply, a strict etymological interpretation would regard lions as antibiotic with respect to humans, and Popeye as antibiotic with respect to spinach. Clearly the word has to be understood in context. 

    In the present discussion this context relates to chemicals which are antibiotic with respect to microorganisms which can invade the body of a host organism, such as man. Antibiotics are not "antiseptics" or "disinfectants" which can sterilize at the surface or outside of a host, but are usually not tolerated within host tissues. 

     Thus, in the present context I would define the noun "antibiotic" as a chemical [of natural or synthetic origin] which, [usually at low concentrations], inhibits microorganisms of some type within a host organism, while not unacceptably interfering with the life of that organism.

[This does not exclude the possibility that the chemical will also inhibit the microorganisms outside the host (e.g. on a Petri dish) but, by virtue  of being tolerated within the body of the host, the chemical is an antibiotic not an antiseptic. Most modern antibiotics work at low concentrations, and are toxic to the host at high concentrations. It is possible that in future we might find a chemical which works only at  high concentrations and these concentrations are not toxic to the host. So concentration should not be in the definition.]

    Since, historically, many of the early antibiotics worked only against bacteria ("antibacterial antibiotics"), it is easily to fall into the trap of thinking that antivirals ("antiviral antibiotics") are different. If we think of them as in a separate, non-antibiotic, category, then the lessons we have learned about antibiotic usage (i.e. mode of use to prevent resistant strains emerging) can easily get disregarded (the point of my Commentary).

    Unfortunately, the Webster's Dictionary (1976) definition has archaic aspects when defining the noun as "a substance produced by a microorganism and able in dilute solution to inhibit or kill another microorganism". It gets it right when referring to the target of an antibiotic as "another microorganism" (virus, bacterium, fungus, protozoan, etc.), but is wrong in postulating that antibiotics are necessarily "produced by a microorganism".

     Yes, historically, many antibiotics were isolated from microorganisms (e.g. penicillin produced by a fungal mould), but also many were synthesized by the chemist (the arsenicals for the bacterium causing syphilis, and the sulphonamides, which were effective against a variety of bacteria). The list of synthetic antibiotics now includes AZT (antiviral antibiotic) and modified forms of penicillin (antibacterial antibiotics). Antibiotic forms initially derived by purification from microorganisms are now being chemically synthesized and further modified.

       This history can be reversed. Among the antibiotics now available only through chemical synthesis, in future we may find some which are synthesized by some organism, perhaps an organism yet to be discovered. Including the source of antibiotics, and/or the type of microorganisms they attack, in the definition, is archaic. Our nomenclature must move with the times in order not to confuse ourselves, our students, and our patients.

Donald Forsdyke

Another gospel: American Heritage Dictionary 1996.

"NOUN:
A substance, such as penicillin or streptomycin, produced by or derived from certain fungi, bacteria, and other organisms, that can destroy or inhibit the growth of other microorganisms. Antibiotics are widely used in the prevention and treatment of infectious diseases.

ADJECTIVE:
1. Of or relating to antibiotics. 2. Of or relating to antibiosis. 3. Destroying life or preventing the inception or continuance of life".

For more on this matter, see:

Bentley, R.& Bennett, J. W. (2003) What is an antibiotic? revisited.  Advances in Applied Microbiology 52, 303-331.

"However, the most worrisome reservoir consists of latently infected resting memory CD4+ T cells carrying integrated HIV-1 DNA. Definitive demonstration of the presence of this form of latency required development of methods for isolating extremely pure populations of resting CD4+ T cells and for demonstrating that a small fraction of these cells contain integrated HIV-1 DNA that is competent for replication if the cells undergo antigen-driven activation. 

    Most of the latent virus in resting CD4+ T cells is found in cells of the memory phenotype. The half-life of this latent reservoir is extremely long (44 months). At this rate, eradication of this reservoir would require over 60 years of treatment. Thus, latently infected resting CD4+ T cells provide a mechanism for life-long persistence of replication-competent forms of HIV-1, rendering unrealistic hopes of virus eradication with current antiretroviral regimens. 

    The extraordinary stability of the reservoir may reflect gradual reseeding by a very low level of ongoing viral replication and/or mechanisms that contribute to the intrinsic stability of the memory T cell compartment. Given the substantial long-term toxicities of current combination therapy regimens, novel approaches to eradicating this latent reservoir are urgently needed."

T. Pierson, J. McArthur & R. F. Siliciano. (2000) Annual Reviews of Immunology 18, 665-708.

"In cases with apparent complete HIV suppression by HAART, viral rebound after cessation of therapy could have originated from the activation of virus from the latent reservoir." (Zhang et al. 2000).

In a major concession to the viewpoint advanced on this web-page it was stated:

"Several research goals assume paramount importance.

• First, it is critical to determine whether additional viral reservoirs exist.

  
  

• Second, it is important to understand the nature and source of the ongoing virus production that is seen in most patients on HAART.

  
  

• Finally, novel approaches are needed to eliminate latently infected cells which clearly represent a very serious barrier to HIV-1 eradication."

That such research goals "assume" importance only by the year 2000 is of note. The readers of these pages may infer that individuals such as the authors cited above were among those rejecting grant applications from researchers who, a decade or more before, had considered it quite obvious that these goals were of paramount importance.

    Their goals did not get support, not because of scientific logic, but because of the mind-set of the well-intentioned people who held the political high ground, but all-too-often could not see beyond their noses. 

    There is nothing new in this, as the example of diphtheria immunization in the early decades of the 20th century has shown (Forsdyke, 2000). Amazingly, in 2003 Dr. Siliciano declared that: "It is difficult to envision any targeting mechanism that will allow specific elimination of this reservoir."(Click Here). Of course, he is not required to "envision." He just has to read the scientific literature (see above)!

     The implications of this go far beyond the management of natural diseases. Currently, the greatest threat to humankind seems to be overt or terrorist warfare conducted not with nuclear weapons, but with biological weapons. A nation which uses the peer-review process, as it currently (2001) operates, to select those who give it advice on biomedical matters, may not fare well in confrontation with a nation which has adapted the peer-review process to identify those (e.g. Irvine Page, Szent-Gyorgyi, Erwin Chargaff) who can see beyond their noses. 

Forsdyke, D. R. (2000) Tomorrow's Cures Today? How to Reform the Health Research System. Harwood Academic, Newark.

Siliciano, J. D. & Siliciano, R. F. (2000) Latency and viral persistence in HIV-1 infection. J. Clin. Invest. 106, 823-824.

Zhang, L., Chung, C., Hu, B-S., He, T., Guo, Y., Kim, A. J., Skulsky, E., Jin, X., Hurley, A., Ramratnam, B., Markowitz, M. & Ho, D. D. (2000) J. Clin. Invest. 106, 839-845.