HIVSuperinfection and Immune Control: Implications for Vaccine Development? Todd M. Allen, phd Instructor in Medicine, Harvard Medical School, Massachusetts General Hospital and Partners aids Research Center Reprinted from The PRN Notebook,™ december 2002. Boston, Massachusetts Dr. James F. Braun, Editor-in-Chief. Tim Horn, Executive Editor. Published in New York City by the Physicians’ Research Network, Inc.,® Summary by Tim Horn John Graham Brown, Executive Director. For further information and other articles prn december 2002 Edited by Frederick M. Hecht, md; Martin Markowitz, md available online, visit http://www. .org All rights reserved. © .

n the last day of the 7th confer- there has been much consternation re- nine of 14 newly infected patients who ence on Retroviruses and Oppor- garding the conclusions drawn from the initiated haart, followed by one or more tunistic Infections in San Francisco Ottawa case report. For starters, the full treatment interruptions, have maintained in February 2000, shockwaves re- details of this case have not been published robust hiv-specific CD4+ and CD8+ cell re- verberated through the Moscone in a peer-reviewed medical journal. It is sponses and low viral loads in the ab- Convention Center. No, it was not an also unclear whether the strain causing sence of treatment; for more than two earthquake attributed to the cantanker- the primary infection, the superinfecting years in some cases. Yet there have also ous San Andreas Fault but rather an earth- strain, or a potential recombinant form been cases where this control has not shattering case report stemming from a had resulted in the patient’s rapid disease proven durable. Some patients have not Canadian hiv clinic situated 2500 miles progression, given that comprehensive ge- been able to maintain appreciable con- away (Angel, 2000). The case report, pre- netic analyses were not conducted. There trol of hiv replication, irrespective of the sented by Dr. Jonathan Angel and his col- has also been some speculation that the number of treatment interruptions at- leagues from the University of Ottawa, in- purported superinfection reported by Dr. tempted. Most intriguing has been one volved an antiretroviral-naive hiv-posi- Angel and his colleagues was a case of lab- subject in particular—a patient who ini- tive male (patient A) who experienced oratory error because of contamination— tially experienced virologic control in re- rapid disease progression and high lev- which speaks more to the challenges of sponse to early therapy and treatment in- els of viral resistance to multiple drugs identifying superinfection than about su- terruptions, only to experience a subse- after engaging in unprotected sexual ac- perinfection itself. quent sudden loss of virologic control and tivity with another hiv-positive male har- There is now fresh evidence from two disease progression. The case report was boring a drug-resistant, possibly more vir- new, heavily deconstructed case reports to first presented by Dr. Bruce Walker of the ulent strain of hiv (patient B). Dr. Angel conclude, beyond a reasonable doubt, that Partners aids Research Center at the xiv concluded that “Patient A was very likely intersubtype and intrasubtype hiv super- International aids Conference (Walker, infected with a resistant strain of HIV-1 by infection can occur. What remain, how- 2002) and will be discussed more fully in Patient B,” and went on to say: “I think ever, are serious concerns surrounding a pending issue of Nature. there’s enough information here to raise these findings—deep-seated anxieties that The individual in question—dubbed awareness regarding hiv superinfection hiv superinfection may have a significant subject AC-06—presented to the Massa- and to say that this should be a public impact on public health initiatives and chusetts General Hospital clinic with symp- health issue if we can prove it.” vaccine development. tomatic acute infection. He was negative hiv superinfection is defined as a sec- for hiv antibodies but had a viral load ap- ond infection, after a primary infection proaching 9 million copies/mL. Four days has been established, with a heterologous after learning of his infection, he initiated strain belonging to the same subtype as A Mystery at Mass General a haart regimen consisting of nelfinavir, the primary strain (intrasubtype superin- as has been reviewed in several past stavudine, and lamivudine. His viral load fection) or to a different subtype (inter- issues of The PRN Notebook, the Partners rapidly decreased to undetectable levels subtype superinfection). aids Research Center at the Massachu- (see Figure 1). While there have been several reports setts General Hospital has been conducting Five hundred forty-six days after begin- demonstrating hiv coinfection—the simul- some extraordinary research involving ning treatment, he initiated his first treat- taneous transmission of two or more hiv the long-term control of hiv viremia with ment interruption. Sixty-three days into his variants—data supporting the possibility the early initiation of antiretroviral thera- first sti, his hiv-rna level exceeded 50,000 of superinfection have been limited, with py in acutely infected patients and subse- copies/mL, meeting requirements for reini- the Ottawa case report being one of the quent supervised treatment interruptions. tiation of therapy. After three and one-half only examples to draw upon. However, There have been numerous successes— months of successful retreatment with the

4 THE PRN NOTEBOOK™ • VOLUME 7, NUMBER 4 • DECEMBER 2002 • WWW.PRN.ORG same haart regimen, a second sti was ini- tiated. Although there was a noticeable burst in viremia two months into the second haart sti, his viral load spontaneously declined, 200000 remaining below 5,000 copies/mL for more virus 1 virus 2 than seven months. Two hundred-ninety days after stop- 150000 ping therapy for the second time—1005 days after initiating treatment—patient AC-06 experienced a sudden rapid increase 100000 (lcopies/ml) in his viral load and a marked decrease in his CD4+ cell count. Consequently, haart 50000 was reinstated for the third time. hiv-rna Once again, subject AC-06’s viral load de- creased to undetectable levels in response 0 to therapy. Four months later, a third sti 0 200 400 600 800 1000 1200 1400 1600 1800 was attempted. This time, there were no signs of immunologic control—his viral Days After Acute Infection load exceeded 50,000 copies/mL within three weeks of stopping treatment. Treat- figure 1. Subject ac-06: hiv-rna Levels During Acute hiv Infection and ment was again restarted, and a fourth sti 60 Months of Follow-Up. was attempted four months later. Although The horizontal bars show the periods during which the patient received highly active an- his viral load did not peak above 50,000 tiretroviral therapy (haart). The horizontal arrows show the change from virus 1 (the first copies/mL, he was unable to keep his viral subtype B virus) to virus 2 (the second subtype B virus). load below 5,000 copies/mL as seen in three sequential blood draws—a secondary Source: Todd Allen, md criteria for restarting therapy. The patient, however, refused therapy and his viral load cus Altfeld and Dr. Xu Yu at the Massa- topes. “This tipped us off and we con- has continued to increase (and his CD4+ chusetts General Hospital. A panel of B ducted phylogenetic analysis of gag se- cell counts decrease) over time. clade overlapping peptides spanning all quences from samples collected during expressed hiv proteins was used to as- acute infection and during the third sti,” sess the broadening of the CD8+ responses elaborated Dr. Allen. “We found that a to patient AC-06’s hiv. Sure enough, hiv-spe- distinct and unrelated second strain of Looking for Answers cific CD8+ cell responses were enhanced hiv had emerged. Testing additional sam- “what we saw with patient ac-06 was during the first and second sti. “The re- ples, we determined that the second virus very different from what we’ve seen in sponses we saw were targeting over two emerged close to the end of the second our other acutely infected patients who dozen distinct ctl epitopes, which really sti, when viremia peaked, and persisted demonstrated signs of virologic control does reflect a broad response to the virus,” through the third sti.” during initial STIs,” Dr. Allen said. “The Dr. Allen explained. However, samples col- Additional analyses demonstrated that virologic control we saw during the first lected during the third sti demonstrated the two viruses—discussed herein as virus- two STIs in patient AC-06 was subsequent- that the epitope-specific CD8+ responses es 1 and 2—varied by only 12% at the ly lost, whereas in our other patients we seen during the first two STIs had changed amino acid level. However, half of the might expect to see prolonged virologic dramatically. “Some of the epitope-spe- known immunologically targeted portions control with each sti. This prompted us to cific responses were maintained and oth- of the two viruses were different. “We evaluate his virus-specific immune re- ers expanded further during the third sti,” were definitely looking at two viruses,” sponses at various time points to look for he said, “but some responses declined, added Dr. Allen. “Both viruses were sub- correlates of immune control loss.” which we couldn’t readily explain.” type B and possessed the R5 phenotype.” Gag-specific CD4+ proliferative re- Could it have been that the patient’s To determine if viruses 1 and 2 estab- sponses were detected during the initial virus mutated faster than epitope-specific lished infection at the same time (coin- round of treatment and the first and sec- CD8+ cell responses could develop? Dr. fection) or if virus 2 established itself after ond treatment interruptions—at least un- Allen’s group documented sequence primary infection with virus 1 (superin- til the sudden increase in viremia, docu- changes with three of the seven targeted fection), Dr. Allen’s group probed further mented 290 days into the second sti. CD8 epitopes located with hiv gag, but it into patient AC-06’s medical records and The next step was to measure virus- was uncertain that changes of this mag- collected samples. Approximately one specific CD8+ cell responses—which are a nitude would result in such a profound month before virus 2 was found, subject critical component of the immune re- loss of immunologic control. Most inter- AC-06 had reported symptoms consistent sponse to hiv—using an interferon-γ en- esting, however, were a significant number with acute retroviral syndrome, including zyme-linked immunospot assay (elispot). of amino acid changes in the flanking re- fever and nightsweats. These symptoms This work was accomplished by Dr. Mar- gions that did not contain CD8+ cell epi- occurred approximately four to eight

THE PRN NOTEBOOK™ • VOLUME 7, NUMBER 4 • DECEMBER 2002 • WWW.PRN.ORG 5 protocol. That was in January of 2001, and by February of 2001 his viral load alvac vcp1452 had rebounded to 80,000 copies/mL and haart then decreased to 21,000 copies/mL. 7 ae b 1600 Blood drawn on April 10, 2001, re- 6 cd4 cell count vealed that the patient had experienced a sharp rebound in viral load, which fluc- 5 1200 tuated between 200,000 and 400,000 4 copies/mL for the next four months. He 800 3 had mild symptoms—including fatigue (log copies/ml) and fever—and lost approximately 300 2 hiv-1 rna 400 CD4+ cells/mm3 during this time. The pa- Cell Count (cells/ml) Cell Count 1 hiv-rna tient noted that he had been in Brazil cd4 0 0 three weeks before the April 10 blood nov march july nov march july nov march july draw and that he had had several unpro- 1998 1998 1998 1999 2000 2000 2000 2001 2001 tected sexual experiences. Four months after this second viral load rebound, the figure 2. The Geneva Subject: hiv-rna Levels and cd4+ Cell Counts During patient consented to restart therapy and his viral load decreased rapidly. Soon after Acute hiv Infection and 31 Months of Follow-Up. restarting treatment for the third time, The horizontal bars show the periods during which the patient received highly active his alanine aminotransferase levels in- haart alvac cp1452 antiretroviral therapy ( ) and the period of vaccination with v , and the creased significantly. Using hcv-pcr, it horizontal arrows show the change from subtype ae to subtype B. was determined that he was in the initial throes of hcv infection. He is now being Source: Jost, 2002. Reprinted with permission of the New England Journal of Medicine and the Massachusetts Medical Society. successfully treated with pegylated inter- feron and ribavirin. weeks after the patient had unprotected team of Swiss investigators. Preliminary A snapshot of this patient’s clinical his- sex with an anonymous partner. pcr test- data from this intriguing report were first tory, beginning with the time of his diag- ing of blood samples revealed virus 1 to be presented at the 9th Conference on Retro- nosis of acute hiv infection to the last date the only detectable virus at the time of viruses and Opportunistic Infections, held of follow-up data reported in the New Eng- acute infection, during the first sti, and for last winter in Seattle, updated at the xiv In- land Journal of Medicine article, is illus- the better part of the second sti. The virus ternational aids Conference this past sum- trated in Figure 2. detected during the rebound in viremia mer in Barcelona, and then published in toward the end of the second sti was September as a brief report in the New found to be virus 2 (see Figure 1). Ana- England Journal of Medicine (Jost, 2002). lyzing viral dna from PBMCs also demon- On November 18, 1998, a 38-year-old What Lies Beneath strated that virus 1 was the only virus de- man presented to the University of Gene- suspecting that a second hiv infection tected through to the rebound in viremia va with an acute retroviral syndrome. An- might be behind the second rebound in during the second sti. From that point tibodies to hiv were not yet detectable, their patient’s viral load, the Geneva forward, both viruses 1 and 2 could be although his p24 antigen titer was greater team—with the help of investigators at detected in PBMCs. than 100 pg/mL and his viral load ex- the Hôpital Pitié Salpêtrière in Paris—se- “All of the data point to hiv superin- ceeded 800,000 copies/mL. quenced the reverse transcriptase genes, fection,” Dr. Allen remarked. “Despite a Soon after receiving a diagnosis of pri- gag p17, and the C2 to V3 region of env of very strong ctl response, this patient was mary hiv infection, the patient enrolled samples collected from November 18, not able to prevent superinfection with a in a clinical trial and received a four-drug 1998, through January 4, 2002. Samples second virus, to which half of the CD8+ regimen consisting of amprenavir, aba- collected in November 1998 and January cell responses remained cross-reactive. cavir, zidovudine, and lamivudine. Twen- and February 2001 indicated that the ae While we did see an induction of new CD8+ ty-one months later, he co-enrolled into hiv subtype was present. In contrast, se- cell responses after superinfection, viremia a therapeutic vaccination trial and was quencing of samples collected in April, wasn’t well contained and the patient’s randomized to receive alvac’s vCP1452. May, and November 2001 and January viral load continued to increase and his Six weeks after beginning haart, his vi- 2002—samples collected after the second CD4+ cells dropped.” ral load dropped to 1,000 copies/mL. Un- rebound in viral load—indicated the pres- fortunately, therapy was proving to be a ence of hiv subtype B. foe to his liver, resulting in therapy dis- To rule out the possibility that their pa- continuation for six weeks. Upon resuming tient had been coinfected with two sub- A Report From Geneva haart, his viral load decreased rapidly, types of hiv at the same time, the research while not specifically discussed by dr. to less than 50 copies/mL. After receiv- team performed plasma pcr with subtype Allen, another noteworthy case of hiv su- ing vCP1452, his antiretroviral therapy was B- and ae-specific primers. Sure enough, perinfection was reported recently by a again halted as per the vaccine research it was determined that the only detectable

6 THE PRN NOTEBOOK™ • VOLUME 7, NUMBER 4 • DECEMBER 2002 • WWW.PRN.ORG virus circulating in the patient’s blood Philip Goulder and Bruce Walker of Mass- On a more positive note, Dr. Allen from March 1998 to March 2001 was of achusetts General Hospital attempt to ad- pointed out that patient AC-06 did generate the ae subtype. In April 2001, when the dress some of the implications hiv super- new CD8+ cell responses against epitopes second rebound in viral load was detected, infection may have on hiv vaccine initia- that had not been previously presented amplicons from both the ae subtype and B tives (Goulder, 2002). While Drs. Goulder after the emergence of the second virus, subtype were detected. and Walker wrote, “there are aspects of which suggests that therapeutic immu- Delving further, the research team in- (the Geneva case report) that need not nization in chronic infection may be pos- vestigated the responses of CD8+ cells to the leave us in despair,” it appears that some sible. “If we are to better understand the patient’s ae- and B-specific epitopes us- of their more optimistic comments are re- obstacles that may face vaccine develop- ing elispot. Only cells directed against futed by their own, more recent hiv su- ment or to develop effective therapeutic subtype ae-specific epitopes were detect- perinfection case report. vaccines, we need to continue studying ed between November 1998 and March For example, the hiv-specific immune immune responses in patients who control 2001. They markedly decreased after the responses that were detected in the Swiss hiv replication and then lose control,” Dr. switch to the B subtype in April 2001. patient were narrowly directed and in- Allen said. “These types of studies should And to make matters worse, none of the substantial, with CTLs targeting only a sin- provide important insights regarding the subtype B epitopes derived from the pa- gle region of the virus identified. The typ- extent and magnitude of hiv-specific im- tient’s sequences were recognized at any ical broadening of immune responses in mune responses that are needed to pro- time during either subtype ae or subtype this patient may have been blunted by vide cross-protective immunity.” B infection. early haart use and may have further waned during the period of effective ther- apy. In other words, the virus-specific im- mune response may simply have been too Superinfection: limited to protect against another virus, The Great Vaccine Debate particularly of a different subtype. References irrefutable data concluding that hiv Turning to the Boston patient, limited Angel JB, Kravcik S, Balaskas E, et al. Documenta- tion of HIV-1 superinfection and acceleration of superinfection can and does occur is only ctl responses did not appear to be a prob- disease progression [Abstract LB-2]. 7th Confer- the tip of the iceberg. What is necessary lem. Prior to the emergence of the second ence on Retroviruses and Opportunistic Infections, now is to determine its clinical significance, virus, over two dozen CD8+ cell epitopes San Francisco, 2000. which could very well spell trouble for both were detectable, indicating a broad im- Goulder PJR, Walker BD. HIV-1 superinfection—a people already infected with hiv and those mune response. Still, such responses were word of caution. N Engl J Med 347(10):757-8, 2002. who could potentially benefit from a pre- not capable of controlling a second subtype Jost S, Bernard M-C, Kaiser L, et al. A patient with ventive hiv vaccine in the future. B virus that varied by only 12%. A central HIV-1 superinfection. N Engl J Med 347(10):731-6, In an editorial appearing in the Sep- question thus remains: just how broad will 2002. tember 5, 2002, issue of the New England vaccine-induced immune responses need to Walker B. Harnessing the Immune System to Fight Journal of Medicine accompanying the be to successfully prevent—or rapidly con- HIV Infection [Abstract WeOrA197]. XIV Interna- Geneva superinfection case report, Drs. trol—primary hiv infection. tional aids Conference, Barcelona, 2002.

notes

THE PRN NOTEBOOK™ • VOLUME 7, NUMBER 4 • DECEMBER 2002 • WWW.PRN.ORG 7