Proc. Natl. Acad. Sci. USA Vol. 93, pp. 3972-3977, April 1996 Medical Sciences Human immunodeficiency type 1 infection despite prior immunization with a recombinant envelope regimen M. JULIANA MCELRATH*, LAWRENCE COREY*tS, PHILIP D. GREENBERG*§, THOMAS J. MATTHEWSI, DAVID C. MONTEFIORI¶, LEE ROWEN*, LEROY HOOD*§II, AND JAMES I. MULLINS*t Departments 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- MATERIALS AND METHODS virus 1 it tive human immunodeficiency type (HIV-1) vaccine, A 24-year-old HIV-1 seronegative, healthy Caucasian who remains unclear which immune responses are sufficient to never received a smallpox vaccine and who reported involve- protect against infection and whether prior HIV-1 immunity ment in a stable monogamous sexual relationship with an can alter the subsequent course of HIV-1 infection. We HIV-uninfected partner was recruited for the study. Over 4 investigated these issues in the context of a volunteer who years, the volunteer received 6 HIV-1 envelope vaccinations received six HIV-1LAI envelope immunizations and 10 weeks (5, 7, 11) as outlined in Table 1. Approximately 3 months (week thereafter acquired HIV-1 infection through a high-risk sex- 206) after the last vac-env booster, the volunteer had an ual exposure. In contrast to nonvaccinated acutely infected increased HIV-1 antibody by ELISA and new Gag antibodies individuals, anamnestic HIV-1-specific B- and T-cell re- by Western blot assay (Table 1). Three months later (week sponses appeared within 3 weeks in this individual, and 222), testing revealed a markedly reactive HIV-1 ELISA and neutralizing antibody preceded CD8+ cytotoxic responses. Western blot assay. Upon questioning retrospectively, the Despite an asymptomatic course and an initial low level of subject disclosed that the previously reported monogamous detectable infectious virus, a progressive CD4+ cell decline relationship had ended and during week 203 the subject had and dysfunction occurred within 2 years. Although vaccina- engaged in unprotected anal intercourse with a new partner of tion elicited immunity to HIV-1 envelope, which was recalled unknown HIV-1 serostatus. Analysis of stored frozen speci- upon HIV-1 exposure, it was insufficient to prevent infection mens revealed no detectable HIV-1 RNA in sera from weeks and subsequent immunodeficiency. 140, 164, 193, 197, and 201, but HIV-1 RNA was detected in serum from week 206 (Table 1). These data indicate that HIV-1 probably was acquired at approximately week 203, The progressive spread of human immunodeficiency virus coincident with the high risk exposure, which we have desig- (HIV) infection has made development of a preventive vaccine nated as week 0 of infection. a global health priority. The immune responses that correlate Individuals with early HIV-1 infection at the University of with protection from HIV infection or control of disease are Washington Center for AIDS Primary Infection Clinic served unknown. Presumably, a vaccine is more likely to be efficacious as the nonvaccinated infected control group. Immune re- if it is capable of eliciting HIV-specific broad neutralizing sponses in these individuals were determined from peripheral antibodies, CD8+ cytotoxic T lymphocytes (CTL), and T blood mononuclear cells (PBMC) at various times following helper responses (1). Several vaccine regimens have provided presumed or known date of seroconversion. protection in nonhuman primates against challenge with HIV Antibody and Cellular Immune Assays. HIV-1 antibody was or simian immunodeficiency virus (SIV) (2-4). These findings measured by EIA (Genetic Systems, Seattle) and by Western have led to human trials to evaluate a variety of HIV-1 blot (Epitope, Beaverton, OR). Neutralizing antibody assays candidate (5-11). were performed as described (10, 11). Titers represent the Since the initiation of HIV vaccine trials by the AIDS reciprocals of sera dilutions required to reduce infectious virus Vaccine Clinical Trails Network, some study participants have titer by one log or >90% in CEM cell lines (for HIV-1LAI and acquired HIV infection, all as a result of high risk behavior HIV-1MN) and in PBMC (for the first isolated autologous (12). This report provides a detailed analysis of HIV-1 infec- HIV-1). C'-ADE was measured in MT-2 cells (13) by p24 tion in a vaccinated volunteer from one of these studies and production and is expressed as the reciprocal of the last serum compares these responses with a group of nonvaccinated dilution to show enhancement (titer), the reciprocal serum received dilution producing the greatest enhancement (peak), and the individuals with early HIV infection. The volunteer magnitude of enhancement at the peak dilution, given as the six vaccinations, three with a live recombinant vaccinia virus fold-increase in p24 production over background (power). containing HIV-1LAI gp160 (vac-env) and three with a bacu- LP responses were measured in vitro to HIV-1 antigens lovirus-derived HIV-lLAI recombinant envelope protein (2-10 gLg/ml) including psoralen-treated, UV-inactivated pu- (rgpl60) (5, 7, 11). This immunization approach has shown rified HIV-1LAI (kindly provided by S.-L Hu, Bristol-Myers/ promise in the macaque model with SIV infection (3) and has Squibb), gpl60 (HIV-1LAI strain, kindly provided by Micro- been particularly effective at inducing both HIV-specific neu- GeneSys, Meriden, CT), and Env 2-3 (HIV-lsF2 strain, kindly tralizing antibody and T-cell responses (7, 8). While it remains provided by K. Steimer, Biocine, Emeryville, CA), as described unclear why the regimen failed to protect this volunteer, the analysis establishes a framework with which to explore future Abbreviations: HIV-1, human immunodeficiency virus type 1; CTL, cases of HIV infection in vaccinated volunteers participating cytotoxic T lymphocytes; SIV, simian immunodeficiency virus; vac- in expanded clinical trials. env, recombinant vaccinia containing HIV-1LAI gpl60; rgpl60, re- combinant HIV-lLAI gpl60; PBMC, peripheral blood mononuclear cells; C'-ADE, complement-mediated antibody-dependent enhance- The publication costs of this article were defrayed in part by page charge ment; LP, lymphoproliferative; S.I., stimulation index; E/T, effector- payment. This article must therefore be hereby marked "advertisement" in to-target ratio; CC, co-culture. accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 3972 Downloaded by guest on October 2, 2021 Medical Sciences: McElrath et al. Proc. Natl. Acad. Sci. USA 93 (1996)

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Ef- < 10,000 copies/ml at 21 and 25 weeks after infection, and were fectors were tested for lysis of autologous Epstein-Barr virus- intermittently detectable between 39,800 and 82,100 copies/ml transformed B lymphoblastoid cell lines infected with recom- over the next 62 weeks. Attempts to isolate HIV-1 from PBMC binant vaccinia expressing either HIV-1LAI Env, Gag, or Pol were unsuccessful until 30 weeks after infection, and subse- and vaccinia wild-type control or pulsed with HIV-1LAI gp160 quent PBMC cultures were intermittently positive (Table 1). at varying E/T up to 50:1 in a standard 4-h chromium release The mean titer of HIV-1 in PBMCs, when positive, averaged assay (7, 14). 16 infectious units per million PBMCs (data not shown). All Virologic Assays. Plasma HIV-1 RNA was determined by PBMC-derived HIV-1 isolates had the nonsyncytium-inducing branched DNA amplification (15). The lower level of sensi- phenotype. Only the last plasma HIV culture attempted at tivity of the assay is 10,000 copies/ml. Infectious HIV-1 was week 103 was positive (Table 1). measured by p24 production from either PBMC or plasma as HIV-1 envelope gene sequences were amplified using PCR, described (16). The ability of the autologous viral isolate to cloned, and sequenced from the volunteer's PBMC taken on induce syncytia was determined in MT-2 cells (17). weeks 5 and 21 after infection, and from a virus culture HIV-1 env sequences were amplified using PCR directly initiated with PBMC drawn on week 30 after infection (two from PBMC obtained at weeks 208 and 224, and after co- clones, designated CCa and CCb). As shown in Fig. 1, for week culture (CC) of the recipient's PBMC drawn from week 233 5 and 30 specimens, these sequences were closely related, with PBMC from an uninfected donor. The fragment from the reflecting their origin from a single infected individual and first PBMC sample was generated by nested PCR (18) using belonged to the B envelope sequence clade, as expected for primers ES7 and ES8 in the second round of PCR, and cloned infections acquired in the United States. They were also into pUC19. The CC sequences were obtained after amplifi- distinct from the HIV-lAI vaccine strain (represented by cation of the entire envelope coding sequence which was then HXB2R in Fig. 1). Two features of note were the presence of cloned into the mammalian vaccinia expression vectors the macrophage-tropic virus consensus sequence in the V3 pWR508 and pJW4303. Each sequence was determined using loop, as expected for recently infected individuals (21-23), and a fluorescence-based Applied Biosystems model 373 auto- the absence of positively charged amino acids at positions 11 mated sequencer using dye-labeled terminator (PBMC sam- and 28 of the loop (R and K residues underlined in Fig. 1), ples) or with dye-labeled primers after random shearing, consistent with the observed absence of syncytium-inducing shotgun cloning, and sequencing of the entire plasmid includ- virus. ing vector (CC clones) (19). Immune Responses After HIV-1 Infection. Neutralizing antibodywas consistently measured in sera collected beginning RESULTS 3 weeks after infection, with neutralization of HIV-1LAI de- tected first at a titer of 1:24 (Table 1). By contrast, sera from Vaccine-Induced Immune Responses. The subject experi- three patients infected within the same year and residing in a enced a primary vaccination response following the initial geographically similar area had low (1:20 titer) to no detect- vac-env, and vaccinia was isolated from swabs (5) taken up to able neutralizing antibody to HIV-1LAI when examined over 14 days from the scarification site. Antibodies to HIV-1 gp160 -1 year of infection (Fig. 2A). These results suggest that after were detected after the second dose of vac-env (Table 1). Four infection, an anamnestic antibody response to the previous weeks after boosting with rgpl60 1 year later, neutralizing immunizing antigen occurred in the vaccinated individual. antibody was demonstrated against HIV-1LAI but not against Neutralizing activity to HIV-1MN was detected at week 19 after the heterologous HIV-1MN and in a retrospective analysis, not infection and rose to 1:965 by week 70 after infection. Titers against the autologous HIV-1 (Table 1, week 64). The vaccine to HIV-1LAI increased to a lesser extent than to HIV-1MN regimen induced strong LP responses to HIV-1LAI and HIV- (Table 1). Neutralizing responses to the first isolated autolo- lsF-2, envelope antigens, with S.I. of >1000 (Table 1). During gous HIV-1 were not demonstrated until after 1 year of the peak response, PBMC transferred into hu-PBL severe infection (Table 1, week 53), and the titers were low (1:15). In combined immunodeficient mice conferred protection in two a retrospective analysis, sera obtained before infection, when of three animals following challenge with HIV-1LAI (20). neutralizing responses to HIV-1LAI were present (week 64), Seven CD4+ clones lysed targets expressing HIV-1LAI gpl60, failed to neutralize the autologous HIV-1 (Table 1). and three of the seven clones also recognized targets express- Because of concern that vaccination may induce infection- ing HIV-1MN gp160 (7). enhancing antibodies which may, upon HIV-1 exposure, in- After 1 year, HIV immunity declined and a third rgpl60 crease the susceptibility to infection or severity of disease (24), boost was administered at week 136. This boost resulted in we retrospectively analyzed stored sera for the presence of increased envelope antibodies, low titer neutralizing antibod- C'-ADE. No enhancing antibodies were detected in any of the ies to HIV-1LAI, but not to HIV-1MN, and an increase in sera tested at six time points prior to the acquisition of HIV-1 lymphoproliferation to homologous HIV-1 antigens (Table 1, (Table 1). C'-ADE were detected at week 3 after infection at week 140). Another vac-env booster was administered at week an endpoint reciprocal dilution titer of 405 and a maximum 193 (Table 1), resulting in a secondary vaccinia reaction. power of enhancement of 11.2, which occurred at the lowest Vaccinia was isolated from the scarification site 4 days after serum dilution tested (1:45) (Table 1). Such antibodies per- inoculation. However, little increase in HIV-1 immunity was sisted but generally at lower levels when measured throughout noted (Table 1). HIV-specific CD8+ CTL, examined for the the subsequent 70 weeks ofinfection (Table 1). Bycontrast, the first time during the vaccine study, were not detected prior to, power of C'-ADE was either low (<4) or undetectable in 7 and at 1 month (weeks 193 and 197) following the third vac-env nonvaccinated individuals with early HIV infection over a immunization (Table 1). 40-week period. Clinical and Virologic Course of HIV Infection. The volun- HIV-1 infection also boosted the LP responses to HIV teer experienced no clinical illness following acute HIV-1 antigens (Table 1). S.I. to HIV-1LAI and gpl60 rose from 1 and exposure, and after 2 years has remained asymptomatic. CD4 58,6 weeks before HIV-1 exposure, to 72 and 357, respectively, counts had fallen from a median of 780 cells/mm3 to 462 21 weeks after infection. These responses, never as high as the cells/mm3 when first diagnosed, and within 98 weeks of peak responses following immunization, were remarkably Downloaded by guest on October 2, 2021 Medical Sciences: McElrath et al. Proc. Natl. Acad. Sci. 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HXB2R ...... L .T ..G...... D ...... N .S ...... C ...... L ...... A ...A CCa VAEGTDRVIEVVQRTYRAILHIPTRIRQGLERALL HXB2R ...... R ...... I.. FIG. 1. HIV-1 envelope sequences in the infected vaccine recipient compared to the vaccine strain HIV-1LAI (HXB2R). HIV-1 env sequences were amplified using PCR directly from a PBMC sample taken -5 weeks after infection (week 208), and from two clones obtained after CC of the recipient's PBMC drawn on week 30 after infection (week 233) with PBMC from an uninfected donor (designated CCa and CCb). Each sequence is compared to that of co-cultured virus "a" with only differences shown in the other sequences. Identical residues are indicated with a dot, and gaps introduced to maintain the alignment are indicated by dashes. The cysteine-bounded V3-loop region is indicated and critical residues referred to in the text are underlined. higher in comparison to 11 HIV-1 unimmunized patients with shortly after infection suggests that prior vaccination primed early HIV infection from 1 to 17 weeks after seroconversion, both memory B and T cells to some HIV-1LAI epitopes, but in whom S.I. to gpl60 ranged from 1 to 12 with a median of that this response was insufficient to protect against infection 1. When examined monthly over the course of 6-12 months of to a different HIV-1 strain and the subsequent development of infection, only two nonvaccinated individuals exhibited an S.I. T-cell immunodeficiency. (iii) The subject had no detectable 24.0 to gpl60 (Fig. 2B). Lymphoproliferation declined in C'-ADE prior to HIV-1 infection, which suggests that vacci- concert with the fall in CD4 counts in the vaccinated subject, nation was unlikelyby this mechanism to increase susceptibility and was of greater magnitude and more durable to HIV-1LAI to infection. envelope (gpl60) than those to the HIV-lsF_2 envelope (ENV The failure of vaccination to protect this individual against 2-3) (Table 1). HIV infection is consistent with recent studies employing a No CD8+ CTL activity against targets expressing HIV-lLAI similar immunization regimen in the nonhuman primate SIV Env or Gag was detected at E/T up to 40:1 after 3 weeks of models. In these investigations, vaccination induced complete infection (Table 1). At week 21, at an E/T of 40:1, lytic activity protection in macaques against a low dose challenge of the of 64% was detected against Gag-expressing (HIV-1LAI strain) homologous biological clone SIV Ells (3), but only partial target cells, but not against Env-expressing (HIV-1LAI or protection against a subsequent high dose challenge with the HIV-lsF-2) target cells. Subsequent evaluations have revealed uncloned parent SIVmne (25). In addition, other investigators intermittent CTL responses to HIV-1 Env and Gag (Table 1), have failed to demonstrate protection with this approach and Pol (data not shown). against SIVmac strains (26-28). Together, these findings strengthen the evidence that protective immunity elicited by this vaccine strategy may be restricted to a narrow antigenic DISCUSSION specificity. Here we present a detailed report of naturally acquired HIV-1 If a vaccine regimen fails to protect against HIV-1 infection, infection in a recipient of the live vector priming and protein a more realistic endpoint may be to slow progression of HIV-1 boosting combination regimen. This approach has elicited disease (24). Indeed, studies in the macaque model have some of the most immunogenic responses in humans among suggested that breakthrough SIV infections following ineffec- currently available subunit envelope-based vaccines (7,8). This tive vaccination regimens (including the prime-boost regi- subject developed strong neutralizing and T-cell responses, mens) have been associated with reduction in viremia and which have been considered essential components in protect- delay in progression to AIDS (27-29). Several reports (30-35) ing against HIV-1 infection (1). Although it is impossible to have identified risk factors in early human infection that may verify which factors contributed to vaccine failure, three correlate with more rapid HIV-1 progression. Our volunteer features should be emphasized. (i) Despite multiple booster demonstrated three characteristics that would not favor rapid injections, the last near the time of HIV-1 exposure, the disease progression: asymptomatic illness, low-level infectious subject was unable to sustain the high levels of HIV-1 immu- virus, and nonsyncytium-inducing phenotype. However, the nity noted after the first year of immunization. Just before individual bears the class II DR1 allele by major histocom- infection with HIV-1, neither detectable neutralizing antibod- patibility complex serologic typing, shown in a cohort of ies to HIV-1 nor HIV-l-specific CD8+ T-cell responses were HIV-1-infected hemophiliacs to be associated with early dis- detectable. (ii) The anamnestic immune response observed ease progression (36). From 3 to 25% of the newly-infected Downloaded by guest on October 2, 2021 3976 Medical Sciences: McElrath et al. Proc. Natl. Acad. Sci. USA 93 (1996) or the of T-cell were insuf- 250 A recognition magnitude responses - vaccinated ficient to contain viral infection in our volunteer is uncertain, T and detailed study of additional cases will be needed to answer , non-vaccinated these I- 200- questions. 01 /0 The appearance of C'-ADE was one of the earliest immune c / .__ responses detected following infection. The C'-ADE in the N 7 vaccinated volunteer's sera was significantly higher than that in - 150 the sera of seven nonvaccinated acute seroconverters, suggest- 1) -0 / ing that previous vaccination with gp160 products may more z induce this after infection, but these results o 100- likely response / await confirmation with a larger sample population. The I significance of antibody enhancement of infection in control- 0 ling plasma viremia or influencing clinical progression after 0) 50 early HIV-1 infection remains to be determined. What have we learned from this lead case that can be applied 10 W% a induction of a <10 ==X 0 0 to vaccine development? First, despite pro- = -1-1111, 4 -. ,, -I .. T r nounced humoral and T-cell immune response by HIV-1 0 10 20 30 40 50 60 70 80 90 100 vaccination, prior immunity may not be sufficient to protect Weeks after Seroconversion against infection or improve the overall course of infection, as is illustrated in this individual. Because of the wide antigenic 3.0 -r B diversity of HIV-1, vaccines must elicit broad immunity that 1 vaccinated will allow recognition of diverse epitopes. Furthermore, the Lr non-vaccinated immunity must be of sufficient duration to protect beyond the 2.54 X period of peak responsiveness, and methods to repeatedly OJ '0 boost the appropriate responses when they are elicited must be defined. the clearest lesson is that until a r:S5 2.0-+ Perhaps highly 0 efficacious HIV-1 vaccine regimen is available, uninfected individuals who participate in clinical trials must continue to 3 11.5 practice risk reduction in order to avoid HIV-1 exposure and E infection. o 11.0 We are indebted to David Berger, Dr. Luwy Musey, Mark Hoffman, 0 Sara Klucking, Mike Rabin, Dr. Stephen Kent, Dr. James Arthos, -J Marta Schulte, Mary Ellen Ahearn, Mike Ankener, Jason Sets, Dale 0.5-+ Baskin, Chetana Acharya, Nancy Coomer, Pam Easterling, and to all the volunteers who have provided their time and dedication in 0.0L the interests of developing an HIV vaccine. This work was supported gp160 ENV 2-3 by National Institutes of Health Grants AI-05056, AI-27757, AI-32885, and AI-15106 and National Science Foundation Grant Envelope Antigen NSF-BIR9214821A001. FIG. 2. Comparison of HIV-1-specific immunological responses 1. Haynes, B. F. (1993) Science 260, 1279-1286. between nonvaccinated individuals and the vaccine recipient following 2. Berman, P. W., Gregory, T. J., Riddle, L., Nakamura, G. R., HIV-1 infection. (A) The reciprocal of serum neutralizing antibody Champe, M. A., Porter, J. P., Wurm, F. M., Hershberg, R. D., titers against HIV-1LAI is shown for the infected patient (0) and for Cobb, E. K. & Eichberg, J. W. (1990) Nature (London) 345, three nonvaccinated HIV-infected patients (0, *, *). (B) Lymphopro- 622-625. liferative responses are depicted to HIV-1LAI gpl60 and HIV-lsF-2 3. Hu, S.-L., Abrams, K., Barber, G. N., Morgan, P., Zarling, J. M., ENV 2-3 in the patient 21 weeks after infection and in 11 nonvacci- Langlois, A. J., Kuller, L., Morton, W. R. & Benveniste, R. E. nated individuals at a mean of 9 weeks after seroconversion (range (1992) Science 255, 456-459. 1-17). Results are expressed as the loglO S.I. 4. Daniel, M. D., Kirchhoff, F., Czajak, S. C., Sehgal, P. K. & will CD4 counts of <300 within Desrosiers, R. C. (1992) Science 258, 1938-1941. individuals with HIV develop 5. Cooney, E. L., Collier, A. C., Greenberg, P. D., Coombs, R. W., 1 to 3 years after infection (37, 38), which is similar to the Zarling, J., Arditti, D. E., Hoffman, M. C., Hu, S.-L. & Corey, L. pattern our volunteer developed. (1991) Lancet 337, 567-572. The immune responses in this previously vaccinated indi- 6. Dolin, R., Graham, B. S., Greenberg, S. B., Tacket, C. O., Belshe, vidual differed from that in nonimmunized individuals with R. B., Midthun, K., Clements, M. L., Gorse, G. J., Horgan, B. W., HIV-1 infection. This individual generated neutralizing anti- Atmar, R. L., Karzon, D. T., Bonnez, W., Fernie, B. F., Monte- to CD8+ CTL the neutraliz- fiori, D. C., Stablein, D. M., Smith, G. E., Koff, W. C. & National body prior responses. Moreover, Institute of and Infectious Diseases AIDS Vaccine was to the not to the Allergy ing activity initially vaccinating strain, Clinical Trials Network (1991) Ann. Intern. Med. 114, 119-127. autologous virus or other more closely related clade B strains. 7. Cooney, E. L., McElrath, M. J., Corey, L., Hu, S.-L., Collier, These findings contrast with those in natural infection in most A. C., Arditti, D., Hoffman, M., Coombs, R. W., Smith, G. E. & nonimmunized patients reported by Koup et al. (39) and as Greenberg, P. D. (1993) Proc. Natl. Acad. Sci. USA 90, 1882- shown in our laboratory (Fig. 2A). Moreover, lymphoprolif- 1886. eration to HIV-1 envelope detected at weeks 3 and 19 after 8. Graham, B. S., Matthews, T. J., Belshe, R. B., Clements, M. L., infection were higher in this individual than that seen in >30 Dolin, R., Wright, P. F., Gorse, G. J., Schwartz, D. H., Keefer, HIV-1-infected individuals with CD4+ T-cell counts of >600 M. C., Bolognesi, D. P., Corey, L., Stablein, D. M., Esterlitz, J. G. P. W. C. & National and in 11 with acute HIV-1 as R., Hu, S.-L., Smith, E., Fast, E., Koff, patients primary infection, Institute of Allergy and Infectious Diseases AIDS Vaccine shown in Fig. 2B. However, in temporal association with a Clinical Trials Network (1993) J. Infect. Dis. 167, 533-537. decline in LP responses to HIV-1 envelope antigens has been 9. Schwartz, D. H., Gorse, G., Clements, M. L., Belshe, R., Izu, A., a progressive fall in CD4+ T-cell counts and the persistent Duliege, A. M., Berman, P., Twaddell, T., Stablein, D., Sposto, detection of infectious virus. Whether the patterns of epitope R., Siliciano, R. & Matthews, T. (1993) Lancet 342, 69-73. Downloaded by guest on October 2, 2021 Mledical Sciences: McEslrath et al. Proc. Natl. Acad. Sci. USA 93 (1996) 3977 10. Belshe, R., Clements, M. 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