Virological and Immunological Aspects of AIDS Pathogenesis

The most common and serious problem associated with long term antiretroviral therapy is waning efficacy over time. To date. a number of studies has suggested an association between drug resistance and clinical deterioration. However. a precise causal relationship has yet to be demonstrated. In a large American clinical trial. resistance to zidovudine (ZDV) was predictive of subsequent disease progression if this therapy was continued. Surprisingly. this was also predictive of deterioration if therapy was changed to didanosine (ddl). This suggests that other factors (perhaps virological and immunological) which may be present in addition to resistance. were as important (if not more so) in predicting clinical outcomes. It is likely that viral load. resistance. viral phenotype and alterations in immune function interact in this regard. Proper· studies may allow us to determine a “threshold” for a composite virological and immunological parameter beyond which disease progression will occur. As more antiretroviral agents become available. we will be in a position to intervene to “improve” laboratory markers and monitor them prospectively. potentially to maintain clinical latency for an indefinite period of time. In the authors' laboratories, a quantitative polymerase chain reaction assay for the evaluation of circulating proviral load has been developed. In an initial study of 70 patients. proviral load/ 106 CD4 cells was clearly associated with the severity of immune disease. with up to 9.6% of cells being infected in subjects with CD4 cell counts below 200/µL. However. large variability in proviral load among individuals with comparable or dissimilar CD4 cell counts precludes the use of this measurement as an individual marker of the severity of immune disease. More recent work evaluated the combined use of proviral load (expressed as a dichotomous variable based on values above or below one copy/a03 CD4 cells) and resistance in a prospective fashion. In five patients with high proviral loads and isolates resistant to their current therapy. a mean decrease of 72 CD4 cells/µL was observed over 12 months of observation. In contrast. in six patients with low proviral loads and sensitive isolates. there was a mean increase of 43 CD4 cells/µL. It appears that virological markers are associated with immune disease progression in this small cohort of patients. The association appears most marked when the two virological parameters are considered together rather than individually. The association is not always a tight one. and this may relate to a number of unmeasured factors. including viral phenotype. plasma viremia. and the immune response to HIV infection. Additional work incorporating these parameters into analysis is currently underway in the authors’ centre.

T HE PATI IOGENESIS OF' IIIV INFECTION LEADING TO 'F'ULL-blown· AIDS follows a very precise sequence of events (1). Following exposure to the virus. there is, in many cases, a period of symptomatic primary infection. which is self-limited. Within a number of weeks, it gives way to a period of clinical latency, which usually lasts many years. The process of viral emergence from latency is poorly understood. but it heralds the development of profound immunodeficiency, with frequent life Lhrealening opportunistic infect.ions. sion will increase our insight with respect lo the events surrounding the emergence from viral latency and assist us in the design of optimal health maintenance strategics for 111v-infecled patients.

VI RAL LOAD MEASUREMENT
Quantitative 111v cultures of plasma and circulating mononuclear cells were first desc1ibcd in 1989 (5). A clear association was noted between viral load and immune disease. It was suggested that. symptoms wou ld develop when a 'threshold' value of 10 3 viruses/ mL of blood was exceeded. In long term prospective follow -up of four patients, this appeared lo remain correct (6). These individuals were clinically stable as long as their circulating viral load remained unchanged. A 1ise in load preceded the development of opportunistic infections, although a precise causal relationship could not be established.
A number of specific parameters has been associated with this observed disease progression (2). These include immunological (increased beta-2 microglobulin and neopterin. and decreased 111v-specific humoral and cellular responses). and virological (increased plasma and cell associated viral load) measures. Conversely, strongly preserved nonspecific and specific immune function and low circu lating and tissue-bound viral load have been associated with clinical stability and long term survival (2,3).
Characteristics associated with increased virulence in the host include an enhanced cellular host range, rapid kinetics of replication and increased syncytium induc ing capacity (4). ll is clear that an increased understand ing of the homeostasis between virological and immunological determinants of disease progres-l4E With the advent of the polyn1erase chain reaction (PCR), a more rapid and powerful tool became available for the measurement of viral load (7). Using a quantitative PCI{ assay developed in our laboratory, we measured circulating proviral load in the mononuclear cells of 70 study patients (8) . Results were expressed as a function of the severity of immune disease. according to CD4 cell count categories (Table 1). us A wide range of proviral loads was observed in each category. Thus we. as others. have shown that proviral load in itself cannot be used for individual disease staging. However. in the groups. a clear associaUon was observed between increasing load and progressive immune disease, wiU1 up lo 9.6% CD4 cells infected in some patients. In addition. when expressing proviral load as a dichotomous variable. we found that in individuals with CD4 cell counts above 500/µL only one of 10 had high circulating proviral loads (more than 10 3 copies/10 6 CD4 cells), compared with 23 of 31 with cell counts below 200/µL (P<0.001). Thus, a threshold-type phenomenon may be present with respect lo lhe clinical significance of this parameter.

ANTIRETROVIRAL RESISTANCE
First developed as an anticancer drug in 1964 (9). zidovudine (zov) was found to have activity against 111v in 1985 (10). Several clinical trials have established the efficacy and toxicity of zov in a variety of HIV-associated conditions. The most common and serious problem associated with long term zov therapy is waning efficacy over lime. The isolation of HIV with reduced susceptibility lo zov was initially reported in 1989 (11).
Generally, there appears lo be a relationship between in vitro resistance and the duration of antiviral therapy. In addition. it appears that resistance occurs more rapidly in patients with advanced HIV infection (12). To dale, a number of studies has suggested an association between zov resistance and clinical or immunological deterioration; however, a precise causal relationship has yet to be demonstrated. In a recent large American study, the baseline prevalence and clinical significance of zov resistance was examined in a group of patients on long term zov therapy who were randomized lo continued zov or a change lo didanosine (ddl) (13). As expected. baseline zov resistance was predictive of clinical deterioration if this therapy was continued. Surprisingly, this was also predictive of deterioration if therapy was changed to ddl. This suggests lhal other factors (perhaps virological and immunological) which were also present in individuals with resistant isolates, were as important (if nol more so) than resistance itself in predicting impending deterioration. ll should be staled that. in this study. the mean CD4 cell count in participating patients was below 50 cells/µL. In addition, a Canadian study of patients with very mild disease (CD4 more than 500 cells/µL) showed a clear association between zov resistance and disease progression while on zov (14). ll is more likely that. in such individuals. the negative effect of zov resistance could have been observed in isolation, in the absence of other confounding variables.
In our centre. we have prospectively followed 31 patients to study the correlation between in vitro susceptibility and clinical outcome, as measured by changes in CD4 cell counts (15). Seventeen of 19 indi- victuals with resistant isolates showed immune disease progression. compared with two of 12 with sensitive isolates. It is interesting lo note that lhe two individuals with resistant isolates and stable disease had CD4 cell counts above 300 cells/µL , and likely lower circulating viral loads. In contrast. the two who progressed with susceptible isolates had CD4 cell counts below 50 cells/µL, and likely higher viral loads. This underscores the probable relationship between at least two virological parameters in the prediction of disease progression.
ADDITIONAL VIROLOGICAL PARAMETERS Recent data suggest that the HIV biological phenotype (defined by the capacity to induce syncylia during co-cultivation with donor lymphocytes) may predict the response to therapy. In one study. one of20 individuals who spent 559 months harbouring a nonsyncyliuminducing (NSI) phenotype progressed lo AIDS, whereas progression was observed in eight of 12 who spent 223 months harbouring a syncylium-inducing SI phenotype (16).
Viral phenotype analysis can be easily incorporated into a model examining multiple virological measures. As such. a group of 32 patients on zov therapy was followed prospectively for a mean duration of 34 months ( 17). Over time. patients· viral isolates could be classified as sensitive or resistant (S/R) lo zov. and NSI/SI. Thus, four groups could be generated according lo these two parameters (Table 2). Patients in the R/ SI group had the most significant decrease in CD4 cell count over the period of observation and the highest circulating proviral load. Conversely, patients with the most favourable virological profile (S/NSI. low proviral load) had lhe most favorable CD4 cell count profile.
In our centre. we identified a cohort of 22 HIV-infected individuals. all of whom had received long term zov therapy and had experienced at least a 30% decline in CD4 cell count since initialing this therapy (18). PaUents either remained on zov therapy or were changed to ddl, according to their physicians' discretion. In prospective follow-up. monthly CD4 cell counts were obtained. In addition , every three months. circulating proviral load was measured by quantitative PCR. Susceptibility to current antirelroviral therapy was also measured at the same point in time.
At entry. there were eight individuals with zov-susceptible isolates and 14 with resistant strains. There  were no observable differences in CD4 cell counts (283 and 289 cells/µL. respectively) . We proceeded to examine changes in C D4 cell counts over 12 months as a function of baseline viro logical measures (Table 3).
When proviral load was expressed as a dichotomous variable. individuals wi th higher loads showed a mean decrease in CD-l cell count (-38 cell shtL), compared with the others (+17 cells/µL) . S imilarly. there appeared to be a relationship between drug susceptibility and deterioration in immune function (Tab le 3) . However. a more striking relationship between virological parameters a nd CD4 cell counts was observed when proviral load and resistance were considered together. Over one year of observation. s ix patients with low proviral loads and susceptible viral strains showed a mean increase of 43 CD4 ce llshtL. In contrast. five patients with high load and res istant strains had a mean decrease of 72 C D4 cellsh1L.
IMMUNOLOGICAL PARAMETERS As staled above. the development of effective antiviral drugs is ha mpered by the rapid emergence of drug resistance and of viral phenotypes that are more pathogenic. In this context. an understanding of the relationship between virological measurements and immune dysfunction may be of importance in the design of a lternative therapeu lie modali ties to avoid I irv disease progression .
Cellular and humoral immune responses lo 111vassocialed antigens develop one to three months after infection (19.20). The relationsh ip between the development of n e utralizing antibodies to enve lope glycoproleins and 111v disease progression remains controversial (21.22). 1 lowever. cyto toxic T lymphocytes specific for 11 1v antigens (23) may confer resistance to I IN disease progression (24) and their activity decreases with clinical deterioration (25). The loss of T helper function precedes and predicts the rate of decline in CD4 cell numbers (26.27). T helper cell s sequentially lose the ability to respond to recall antigens. alloantigens and finally to m itogens (28). Alloantigen and mitogen responses improve on antiretroviral therapy. but not the responses lo recall antigens. Because these latter responses are dependent on memory cells. and there is a 16E Changes in CD4 cell count over 12 months of observation , expressed as a function of proviral load and drug susceptibility, considered individually or as a composite virological parameter n Proviral load (copies/ l 0 6 CD4 cells) ~ l 000 (high) 9 < 1000 (low) 13 Drug susceptibility selective depletion of memory cells in IIN infection. improved T helper function may be more easily achieved for responses that arc less dependent. on these cell s (29) . There is lit.lie effect on responses involving a ntigen presentation by monocyles. The observed improvement in T helper function seen in some pati ents may correlate with clinical stab ility in the absence of any changes in CD4 cell counts (30). which sugges ts the need to include T helper function tests as markers of disease progression. Monocytes synthesize a cytokinc. inlerleukin -12 (IL 12). which induces T helper function towards a strong cell -mediated response. It also has an inhibitory effect towards humoral immune responses. Replacement of IL-12 in vitro restores antigen recall functions in peripheral blood lymphocytes of 111v-seropositivc patients. but it does not enhance recall responses in Hrv-scro n egative individuals. 11,-12 deficiency may play an initial role in immunopathogenesis or 111v infect.ion (3 1).
The role of lymphoid organs in th e pathogenesis of 111v infection has recently been investigated (32.33). Following primary infection , 111v lends to localize more in the lyn1phoid organs than in the pe1 ipheraJ lymphocytes. The number of cells harboring I IN DNA and RNA is five to 10 times higher in lyn1ph nodes than in peripheral blood lymphocytes (32), and during periods of clinical latency, most of the 111v load is carried in lyn1phoid organs (34). Follicu lar dendritic cells may be a major site for HN replication and a source of circu lating CD4 cell infection (33). In contrast to monocytes , which circulate in the blood compartment for one day. lymphocytes migrate every 30 mins , on average. from the blood lo lymphoid and nonlymphoid organs and back to the blood via the lymphatics (35) . Ilence. lymphocyte traffic and the close proximity of infected fo llicular dendrilic cells with CD4 cell s suggests that the opportunity for C D4 cells lo become infected with 111v is greater USE in lymphoid organs than in lhe blood comparlmenl.
As a higher proportion of CD4 cells in lymph nodes is aclivaled (25 lo 50%, compared wilh 5 t.o 10% in the circu lalion) (34). lhey are more susceptible lo 111v infection. This may also expla in lhe higher relative viral load lhal may be present in lhis compartment. This may be aided by a functional absence of cyloloxic T cell aclivily in lymph nodes. as previously demonstrated in simian immunodeficiency virus-infected macacques (36) . Thus, a comprehens ive model ofvirological and immunological aspects of AIDS pathogenesis will have lo consider even ls al I.he level of the lymph node.

A UNIFYING HYPOTHESIS
Although our work is far from complete. our preliminary results can be placed wit.bin a general hypothesis of 111v disease progression. An infected individu al begins wilh a given viral load and is placed on antiviral therapy. Over lime, I.he viral stra ins remain susceptible lo I.he agenl being used. or may d evelop resistance. If susceplibilily is maintained in I.he cont.ext. of a relatively low viral burden. immune disease will probably be stable. If, however. viral load shou ld increase. I.he palienl may develop more severe immune disease. A number of fact.ors may serve to increase viral load. These include a change in viral phenotype towards more rapidly replicating s1 stra in s. Once resistance develops. il may be lhal clinical disease will remain stable in lhe selling of a low circu lating viral load. Our challenge in lhis context is to intervene to maintain susceptibility. low viral load and therapeutic efficacy. It is quile clear that no single viral or immune parameter will allow us LY·DONOT cal and immunological aspect of AIDS lo generate dala with optimal clinical significance. A composite virological parameter will have to be generated . including measures of cellular proviral load. vira l phenotype and antiviral susceptibility and T helper cell function. As new RN/\ PCH assays (37) for lhc measurement of circu lating vira l load are standardized and made avail able in retrovirology laboratories. lhey shou ld be included in this model. We feel thal a proper slalislical analysis of these measurements will allow us lo delerrnine a 'threshold' beyond which disease progression will occur if we do not intervene.
CONCLUSION Recent work suggests that many nonvirological factors may contribute to disease progress ion (38). These include the occurrence of opportun istic infections in and or themselves (39). Certain pathogens have been shown lo up-regulate viral replication directly. A weakening of the hosts· spec ific and nonspecific immune response. by mechanisms thal have not been completely elu cidated , may also lead lo an increased circulating viral burden. Prophylaxis and prompt treatment of opportunis ti c infections (and immun otherapy. when feasible modalities become available) provide us wilh key intervention points to control disease progression. Over the coming years. we expect a number of immune modulators and novel anlirelroviral agents to become ava ilable . With prospective monitoring of composite virological markers and T helper cell function. we will be in a position lo intervene lo 'improve· lhc marker profile. potentially to maintain clinical latency for an indefinite period of lime.