Proc. Natl. Acad. Sci. USAVol. 93, pp. 3972-3977, April 1996Medical Sciences

Human immunodeficiency virus type 1 infection despite priorimmunization with a recombinant envelope vaccine regimenM. JULIANA MCELRATH*, LAWRENCE COREY*tS, PHILIP D. GREENBERG*§, THOMAS J. MATTHEWSI,DAVID C. MONTEFIORI¶, LEE ROWEN*, LEROY HOOD*§II, AND JAMES I. MULLINS*tDepartments of *Medicine, tLaboratory Medicine, §Immunology, lMolecular Biotechnology, and tMicrobiology, University of Washington School of Medicine,Seattle, WA 98195; and IDepartment of Surgery, Duke University Medical Center, Durham, NC 27710

Contributed by Leroy Hood, November 6, 1995

ABSTRACT With efforts underway to develop a preven-tive human immunodeficiency virus type 1 (HIV-1) vaccine, itremains unclear which immune responses are sufficient toprotect against infection and whether prior HIV-1 immunitycan alter the subsequent course of HIV-1 infection. Weinvestigated these issues in the context of a volunteer whoreceived six HIV-1LAI envelope immunizations and 10 weeksthereafter acquired HIV-1 infection through a high-risk sex-ual exposure. In contrast to nonvaccinated acutely infectedindividuals, anamnestic HIV-1-specific B- and T-cell re-sponses appeared within 3 weeks in this individual, andneutralizing antibody preceded CD8+ cytotoxic responses.Despite an asymptomatic course and an initial low level ofdetectable infectious virus, a progressive CD4+ cell declineand dysfunction occurred within 2 years. Although vaccina-tion elicited immunity to HIV-1 envelope, which was recalledupon HIV-1 exposure, it was insufficient to prevent infectionand subsequent immunodeficiency.

The progressive spread of human immunodeficiency virus(HIV) infection has made development of a preventive vaccinea global health priority. The immune responses that correlatewith protection from HIV infection or control of disease areunknown. Presumably, a vaccine is more likely to be efficaciousif it is capable of eliciting HIV-specific broad neutralizingantibodies, CD8+ cytotoxic T lymphocytes (CTL), and Thelper responses (1). Several vaccine regimens have providedprotection in nonhuman primates against challenge with HIVor simian immunodeficiency virus (SIV) (2-4). These findingshave led to human trials to evaluate a variety of HIV-1candidate vaccines (5-11).

Since the initiation of HIV vaccine trials by the AIDSVaccine Clinical Trails Network, some study participants haveacquired HIV infection, all as a result of high risk behavior(12). This report provides a detailed analysis of HIV-1 infec-tion in a vaccinated volunteer from one of these studies andcompares these responses with a group of nonvaccinatedindividuals with early HIV infection. The volunteer receivedsix vaccinations, three with a live recombinant vaccinia viruscontaining HIV-1LAI gp160 (vac-env) and three with a bacu-lovirus-derived HIV-lLAI recombinant envelope protein(rgpl60) (5, 7, 11). This immunization approach has shownpromise in the macaque model with SIV infection (3) and hasbeen particularly effective at inducing both HIV-specific neu-tralizing antibody and T-cell responses (7, 8). While it remainsunclear why the regimen failed to protect this volunteer, theanalysis establishes a framework with which to explore futurecases of HIV infection in vaccinated volunteers participatingin expanded clinical trials.

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

MATERIALS AND METHODSA 24-year-old HIV-1 seronegative, healthy Caucasian whonever received a smallpox vaccine and who reported involve-ment in a stable monogamous sexual relationship with anHIV-uninfected partner was recruited for the study. Over 4years, the volunteer received 6 HIV-1 envelope vaccinations(5, 7, 11) as outlined in Table 1. Approximately 3 months (week206) after the last vac-env booster, the volunteer had anincreased HIV-1 antibody by ELISA and new Gag antibodiesby Western blot assay (Table 1). Three months later (week222), testing revealed a markedly reactive HIV-1 ELISA andWestern blot assay. Upon questioning retrospectively, thesubject disclosed that the previously reported monogamousrelationship had ended and during week 203 the subject hadengaged in unprotected anal intercourse with a new partner ofunknown HIV-1 serostatus. Analysis of stored frozen speci-mens revealed no detectable HIV-1 RNA in sera from weeks140, 164, 193, 197, and 201, but HIV-1 RNA was detected inserum from week 206 (Table 1). These data indicate thatHIV-1 probably was acquired at approximately week 203,coincident with the high risk exposure, which we have desig-nated as week 0 of infection.

Individuals with early HIV-1 infection at the University ofWashington Center for AIDS Primary Infection Clinic servedas the nonvaccinated infected control group. Immune re-sponses in these individuals were determined from peripheralblood mononuclear cells (PBMC) at various times followingpresumed or known date of seroconversion.Antibody and Cellular Immune Assays. HIV-1 antibody was

measured by EIA (Genetic Systems, Seattle) and by Westernblot (Epitope, Beaverton, OR). Neutralizing antibody assayswere performed as described (10, 11). Titers represent thereciprocals of sera dilutions required to reduce infectious virustiter by one log or >90% in CEM cell lines (for HIV-1LAI andHIV-1MN) and in PBMC (for the first isolated autologousHIV-1). C'-ADE was measured in MT-2 cells (13) by p24production and is expressed as the reciprocal of the last serumdilution to show enhancement (titer), the reciprocal serumdilution producing the greatest enhancement (peak), and themagnitude of enhancement at the peak dilution, given as thefold-increase in p24 production over background (power).LP responses were measured in vitro to HIV-1 antigens

(2-10 gLg/ml) including psoralen-treated, UV-inactivated pu-rified HIV-1LAI (kindly provided by S.-L Hu, Bristol-Myers/Squibb), gpl60 (HIV-1LAI strain, kindly provided by Micro-GeneSys, Meriden, CT), and Env 2-3 (HIV-lsF2 strain, kindlyprovided by K. Steimer, Biocine, Emeryville, CA), as described

Abbreviations: HIV-1, human immunodeficiency virus type 1; CTL,cytotoxic T lymphocytes; SIV, simian immunodeficiency virus; vac-env, recombinant vaccinia containing HIV-1LAI gpl60; rgpl60, re-combinant HIV-lLAI gpl60; PBMC, peripheral blood mononuclearcells; C'-ADE, complement-mediated antibody-dependent enhance-ment; LP, lymphoproliferative; S.I., stimulation index; E/T, effector-to-target ratio; CC, co-culture.tTo whom reprint requests should be addressed.

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(5, 7). A stimulation index (S.I.) of -3 was considered positivefor the HIV-1LAI antigen and -4.0 was considered positive forgp160 and ENV 2-3 antigens. For analysis ofCD8+ CTL, freshPBMC were stimulated for two 1-week intervals with autolo-gous HIV-1Ba-L-infected macrophages as described (14). Ef-fectors were tested for lysis of autologous Epstein-Barr virus-transformed B lymphoblastoid cell lines infected with recom-binant vaccinia expressing either HIV-1LAI Env, Gag, or Poland vaccinia wild-type control or pulsed with HIV-1LAI gp160at varying E/T up to 50:1 in a standard 4-h chromium releaseassay (7, 14).

Virologic Assays. Plasma HIV-1 RNA was determined bybranched DNA amplification (15). The lower level of sensi-tivity of the assay is 10,000 copies/ml. Infectious HIV-1 wasmeasured by p24 production from either PBMC or plasma asdescribed (16). The ability of the autologous viral isolate toinduce syncytia was determined in MT-2 cells (17).HIV-1 env sequences were amplified using PCR directly

from PBMC obtained at weeks 208 and 224, and after co-culture (CC) of the recipient's PBMC drawn from week 233with PBMC from an uninfected donor. The fragment from thefirst PBMC sample was generated by nested PCR (18) usingprimers ES7 and ES8 in the second round of PCR, and clonedinto pUC19. The CC sequences were obtained after amplifi-cation of the entire envelope coding sequence which was thencloned into the mammalian vaccinia expression vectorspWR508 and pJW4303. Each sequence was determined usinga fluorescence-based Applied Biosystems model 373 auto-mated sequencer using dye-labeled terminator (PBMC sam-ples) or with dye-labeled primers after random shearing,shotgun cloning, and sequencing of the entire plasmid includ-ing vector (CC clones) (19).

RESULTSVaccine-Induced Immune Responses. The subject experi-

enced a primary vaccination response following the initialvac-env, and vaccinia was isolated from swabs (5) taken up to14 days from the scarification site. Antibodies to HIV-1 gp160were detected after the second dose of vac-env (Table 1). Fourweeks after boosting with rgpl60 1 year later, neutralizingantibody was demonstrated against HIV-1LAI but not againstthe heterologous HIV-1MN and in a retrospective analysis, notagainst the autologous HIV-1 (Table 1, week 64). The vaccineregimen induced strong LP responses to HIV-1LAI and HIV-lsF-2, envelope antigens, with S.I. of >1000 (Table 1). Duringthe peak response, PBMC transferred into hu-PBL severecombined immunodeficient mice conferred protection in twoof three animals following challenge with HIV-1LAI (20).Seven CD4+ clones lysed targets expressing HIV-1LAI gpl60,and three of the seven clones also recognized targets express-ing HIV-1MN gp160 (7).

After 1 year, HIV immunity declined and a third rgpl60boost was administered at week 136. This boost resulted inincreased envelope antibodies, low titer neutralizing antibod-ies to HIV-1LAI, but not to HIV-1MN, and an increase inlymphoproliferation to homologous HIV-1 antigens (Table 1,week 140). Another vac-env booster was administered at week193 (Table 1), resulting in a secondary vaccinia reaction.Vaccinia was isolated from the scarification site 4 days afterinoculation. However, little increase in HIV-1 immunity wasnoted (Table 1). HIV-specific CD8+ CTL, examined for thefirst time during the vaccine study, were not detected prior to,and at 1 month (weeks 193 and 197) following the third vac-envimmunization (Table 1).

Clinical and Virologic Course of HIV Infection. The volun-teer experienced no clinical illness following acute HIV-1exposure, and after 2 years has remained asymptomatic. CD4counts had fallen from a median of 780 cells/mm3 to 462cells/mm3 when first diagnosed, and within 98 weeks of

infection, the counts declined to 255 cells/mm3 and remained-300 cells/mm3 through week 116 after infection.At week 206, 3 weeks after infection, 53,400 copies/ml of

HIV RNA were detected in serum. Levels in plasma fell to< 10,000 copies/ml at 21 and 25 weeks after infection, and wereintermittently detectable between 39,800 and 82,100 copies/mlover the next 62 weeks. Attempts to isolate HIV-1 from PBMCwere unsuccessful until 30 weeks after infection, and subse-quent PBMC cultures were intermittently positive (Table 1).The mean titer of HIV-1 in PBMCs, when positive, averaged16 infectious units per million PBMCs (data not shown). AllPBMC-derived HIV-1 isolates had the nonsyncytium-inducingphenotype. Only the last plasma HIV culture attempted atweek 103 was positive (Table 1).HIV-1 envelope gene sequences were amplified using PCR,

cloned, and sequenced from the volunteer's PBMC taken onweeks 5 and 21 after infection, and from a virus cultureinitiated with PBMC drawn on week 30 after infection (twoclones, designated CCa and CCb). As shown in Fig. 1, for week5 and 30 specimens, these sequences were closely related,reflecting their origin from a single infected individual andbelonged to the B envelope sequence clade, as expected forinfections acquired in the United States. They were alsodistinct from the HIV-lAI vaccine strain (represented byHXB2R in Fig. 1). Two features of note were the presence ofthe macrophage-tropic virus consensus sequence in the V3loop, as expected for recently infected individuals (21-23), andthe absence of positively charged amino acids at positions 11and 28 of the loop (R and K residues underlined in Fig. 1),consistent with the observed absence of syncytium-inducingvirus.Immune Responses After HIV-1 Infection. Neutralizing

antibodywas consistently measured in sera collected beginning3 weeks after infection, with neutralization of HIV-1LAI de-tected first at a titer of 1:24 (Table 1). By contrast, sera fromthree patients infected within the same year and residing in ageographically similar area had low (1:20 titer) to no detect-able neutralizing antibody to HIV-1LAI when examined over-1 year of infection (Fig. 2A). These results suggest that afterinfection, an anamnestic antibody response to the previousimmunizing antigen occurred in the vaccinated individual.Neutralizing activity to HIV-1MN was detected at week 19 afterinfection and rose to 1:965 by week 70 after infection. Titersto HIV-1LAI increased to a lesser extent than to HIV-1MN(Table 1). Neutralizing responses to the first isolated autolo-gous HIV-1 were not demonstrated until after 1 year ofinfection (Table 1, week 53), and the titers were low (1:15). Ina retrospective analysis, sera obtained before infection, whenneutralizing responses to HIV-1LAI were present (week 64),failed to neutralize the autologous HIV-1 (Table 1).

Because of concern that vaccination may induce infection-enhancing antibodies which may, upon HIV-1 exposure, in-crease the susceptibility to infection or severity of disease (24),we retrospectively analyzed stored sera for the presence ofC'-ADE. No enhancing antibodies were detected in any of thesera tested at six time points prior to the acquisition of HIV-1(Table 1). C'-ADE were detected at week 3 after infection atan endpoint reciprocal dilution titer of 405 and a maximumpower of enhancement of 11.2, which occurred at the lowestserum dilution tested (1:45) (Table 1). Such antibodies per-sisted but generally at lower levels when measured throughoutthe subsequent 70 weeks of infection (Table 1). By contrast, thepower of C'-ADE was either low (

Proc. Natl. Acad. Sci. USA 93 (1996) 3975

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FIG. 1. HIV-1 envelope sequences in the infected vaccine recipient compared to the vaccine strain HIV-1LAI (HXB2R). HIV-1 env sequenceswere amplified using PCR directly from a PBMC sample taken -5 weeks after infection (week 208), and from two clones obtained after CC ofthe recipient's PBMC drawn on week 30 after infection (week 233) with PBMC from an uninfected donor (designated CCa and CCb). Each sequenceis compared to that of co-cultured virus "a" with only differences shown in the other sequences. Identical residues are indicated with a dot, andgaps introduced to maintain the alignment are indicated by dashes. The cysteine-bounded V3-loop region is indicated and critical residues referredto in the text are underlined.

higher in comparison to 11 HIV-1 unimmunized patients withearly HIV infection from 1 to 17 weeks after seroconversion,in whom S.I. to gpl60 ranged from 1 to 12 with a median of1. When examined monthly over the course of 6-12 months ofinfection, only two nonvaccinated individuals exhibited an S.I.24.0 to gpl60 (Fig. 2B). Lymphoproliferation declined inconcert with the fall in CD4 counts in the vaccinated subject,and was of greater magnitude and more durable to HIV-1LAIenvelope (gpl60) than those to the HIV-lsF_2 envelope (ENV2-3) (Table 1).No CD8+ CTL activity against targets expressing HIV-lLAI

Env or Gag was detected at E/T up to 40:1 after 3 weeks ofinfection (Table 1). At week 21, at an E/T of 40:1, lytic activityof 64% was detected against Gag-expressing (HIV-1LAI strain)target cells, but not against Env-expressing (HIV-1LAI orHIV-lsF-2) target cells. Subsequent evaluations have revealedintermittent CTL responses to HIV-1 Env and Gag (Table 1),and Pol (data not shown).

DISCUSSIONHere we present a detailed report of naturally acquired HIV-1infection in a recipient of the live vector priming and proteinboosting combination regimen. This approach has elicitedsome of the most immunogenic responses in humans amongcurrently available subunit envelope-based vaccines (7, 8). Thissubject developed strong neutralizing and T-cell responses,which have been considered essential components in protect-ing against HIV-1 infection (1). Although it is impossible toverify which factors contributed to vaccine failure, threefeatures should be emphasized. (i) Despite multiple boosterinjections, the last near the time of HIV-1 exposure, thesubject was unable to sustain the high levels of HIV-1 immu-nity noted after the first year of immunization. Just beforeinfection with HIV-1, neither detectable neutralizing antibod-ies to HIV-1 nor HIV-l-specific CD8+ T-cell responses weredetectable. (ii) The anamnestic immune response observed

shortly after infection suggests that prior vaccination primedboth memory B and T cells to some HIV-1LAI epitopes, butthat this response was insufficient to protect against infectionto a different HIV-1 strain and the subsequent development ofT-cell immunodeficiency. (iii) The subject had no detectableC'-ADE prior to HIV-1 infection, which suggests that vacci-nation was unlikely by this mechanism to increase susceptibilityto infection.The failure of vaccination to protect this individual against

HIV infection is consistent with recent studies employing asimilar immunization regimen in the nonhuman primate SIVmodels. In these investigations, vaccination induced completeprotection in macaques against a low dose challenge of thehomologous biological clone SIV Ells (3), but only partialprotection against a subsequent high dose challenge with theuncloned parent SIVmne (25). In addition, other investigatorshave failed to demonstrate protection with this approachagainst SIVmac strains (26-28). Together, these findingsstrengthen the evidence that protective immunity elicited bythis vaccine strategy may be restricted to a narrow antigenicspecificity.

If a vaccine regimen fails to protect against HIV-1 infection,a more realistic endpoint may be to slow progression of HIV-1disease (24). Indeed, studies in the macaque model havesuggested that breakthrough SIV infections following ineffec-tive vaccination regimens (including the prime-boost regi-mens) have been associated with reduction in viremia anddelay in progression to AIDS (27-29). Several reports (30-35)have identified risk factors in early human infection that maycorrelate with more rapid HIV-1 progression. Our volunteerdemonstrated three characteristics that would not favor rapiddisease progression: asymptomatic illness, low-level infectiousvirus, and nonsyncytium-inducing phenotype. However, theindividual bears the class II DR1 allele by major histocom-patibility complex serologic typing, shown in a cohort ofHIV-1-infected hemophiliacs to be associated with early dis-ease progression (36). From 3 to 25% of the newly-infected

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recognition or the magnitude of T-cell responses were insuf-ficient to contain viral infection in our volunteer is uncertain,and detailed study of additional cases will be needed to answerthese questions.The appearance of C'-ADE was one of the earliest immune

responses detected following infection. The C'-ADE in thevaccinated volunteer's sera was significantly higher than that inthe sera of seven nonvaccinated acute seroconverters, suggest-ing that previous vaccination with gp160 products may morelikely induce this response after infection, but these resultsawait confirmation with a larger sample population. Thesignificance of antibody enhancement of infection in control-ling plasma viremia or influencing clinical progression afterearly HIV-1 infection remains to be determined.What have we learned from this lead case that can be applied

to vaccine development? First, despite induction of a pro-nounced humoral and T-cell immune response by HIV-1vaccination, prior immunity may not be sufficient to protectagainst infection or improve the overall course of infection, asis illustrated in this individual. Because of the wide antigenicdiversity of HIV-1, vaccines must elicit broad immunity thatwill allow recognition of diverse epitopes. Furthermore, theimmunity must be of sufficient duration to protect beyond theperiod of peak responsiveness, and methods to repeatedlyboost the appropriate responses when they are elicited must bedefined. Perhaps the clearest lesson is that until a highlyefficacious HIV-1 vaccine regimen is available, uninfectedindividuals who participate in clinical trials must continue topractice risk reduction in order to avoid HIV-1 exposure andinfection.

We are indebted to David Berger, Dr. Luwy Musey, Mark Hoffman,Sara Klucking, Mike Rabin, Dr. Stephen Kent, Dr. James Arthos,Marta Schulte, Mary Ellen Ahearn, Mike Ankener, Jason Sets, DaleBaskin, Chetana Acharya, Nancy Coomer, Pam Easterling, and toall the volunteers who have provided their time and dedication inthe interests of developing an HIV vaccine. This work was supportedby National Institutes of Health Grants AI-05056, AI-27757,AI-32885, and AI-15106 and National Science Foundation GrantNSF-BIR9214821A001.

FIG. 2. Comparison of HIV-1-specific immunological responsesbetween nonvaccinated individuals and the vaccine recipient followingHIV-1 infection. (A) The reciprocal of serum neutralizing antibodytiters against HIV-1LAI is shown for the infected patient (0) and forthree nonvaccinated HIV-infected patients (0, *, *). (B) Lymphopro-liferative responses are depicted to HIV-1LAI gpl60 and HIV-lsF-2ENV 2-3 in the patient 21 weeks after infection and in 11 nonvacci-nated individuals at a mean of 9 weeks after seroconversion (range1-17). Results are expressed as the loglO S.I.

individuals with HIV will develop CD4 counts of 30HIV-1-infected individuals with CD4+ T-cell counts of >600and in 11 patients with acute primary HIV-1 infection, asshown in Fig. 2B. However, in temporal association with adecline in LP responses to HIV-1 envelope antigens has beena progressive fall in CD4+ T-cell counts and the persistentdetection of infectious virus. Whether the patterns of epitope

1. Haynes, B. F. (1993) Science 260, 1279-1286.2. Berman, P. W., Gregory, T. J., Riddle, L., Nakamura, G. R.,

Champe, M. A., Porter, J. P., Wurm, F. M., Hershberg, R. D.,Cobb, E. K. & Eichberg, J. W. (1990) Nature (London) 345,622-625.

3. Hu, S.-L., Abrams, K., Barber, G. N., Morgan, P., Zarling, J. M.,Langlois, A. J., Kuller, L., Morton, W. R. & Benveniste, R. E.(1992) Science 255, 456-459.

4. Daniel, M. D., Kirchhoff, F., Czajak, S. C., Sehgal, P. K. &Desrosiers, R. C. (1992) Science 258, 1938-1941.

5. Cooney, E. L., Collier, A. C., Greenberg, P. D., Coombs, R. W.,Zarling, J., Arditti, D. E., Hoffman, M. C., Hu, S.-L. & Corey, L.(1991) Lancet 337, 567-572.

6. Dolin, R., Graham, B. S., Greenberg, S. B., Tacket, C. O., Belshe,R. B., Midthun, K., Clements, M. L., Gorse, G. J., Horgan, B. W.,Atmar, R. L., Karzon, D. T., Bonnez, W., Fernie, B. F., Monte-fiori, D. C., Stablein, D. M., Smith, G. E., Koff, W. C. & NationalInstitute of Allergy and Infectious Diseases AIDS VaccineClinical Trials Network (1991) Ann. Intern. Med. 114, 119-127.

7. Cooney, E. L., McElrath, M. J., Corey, L., Hu, S.-L., Collier,A. C., Arditti, D., Hoffman, M., Coombs, R. W., Smith, G. E. &Greenberg, P. D. (1993) Proc. Natl. Acad. Sci. USA 90, 1882-1886.

8. Graham, B. S., Matthews, T. J., Belshe, R. B., Clements, M. L.,Dolin, R., Wright, P. F., Gorse, G. J., Schwartz, D. H., Keefer,M. C., Bolognesi, D. P., Corey, L., Stablein, D. M., Esterlitz,J. R., Hu, S.-L., Smith, G. E., Fast, P. E., Koff, W. C. & NationalInstitute of Allergy and Infectious Diseases AIDS VaccineClinical Trials Network (1993) J. Infect. Dis. 167, 533-537.

9. Schwartz, D. H., Gorse, G., Clements, M. L., Belshe, R., Izu, A.,Duliege, A. M., Berman, P., Twaddell, T., Stablein, D., Sposto,R., Siliciano, R. & Matthews, T. (1993) Lancet 342, 69-73.

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Proc. Natl. Acad. Sci. USA 93 (1996) 3977

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