Saturday 9 March 1991

No 8741

ORIGINAL ARTICLES Safety of and immunological

response to

a

recombinant vaccinia virus vaccine expressing HIV

envelope glycoprotein

In a randomised phase I trial of a recombinant vaccinia virus vaccine expressing the gp160 envelope gene of the human immunodeficiency virus (HIVAC-1e) 35 healthy, HIV-seronegative males, 31 of whom had a history of smallpox immunisation and 4 of whom were vaccinia naive, were vaccinated and then boosted 8 weeks later with HIVAC-1e or standard NY strain vaccinia virus. The frequency, duration, and titre of virus isolation from the vaccination site and occurrence of local sideeffects were similar between the two groups of vaccinees. Vaccinia-naive (vac-n) subjects shed virus from the vaccination site for longer and at a higher titre than did vaccinia-primed (vac-p) individuals (19 vs 7 days and 107 vs 105 pfu/ml, respectively). In-vitro T-cell proliferative responses to one or more HIV antigen preparations developed in 13 of 16 vaccinia-primed subjects inoculated with HIVAC-1e. T-cell responses were, however,

transient and in no subject did antibodies to HIV become detectable. The 2 vaccinia-naive subjects vaccinated with HIVAC-1e showed strong T-cell responses to homologous and heterologous strains of whole virus and to recombinant gp160 protein that remained detectable for over a year; antibodies in both. to HIV envelope also developed Recombinant vaccinia virus vaccines induce T-cell priming to the foreign gene products in most individuals. If used as the sole immunising agent they will be most efficacious in vaccinia-naive individuals.

recombinant vaccinia viruses expressing HIV genes are attractive HIV vaccine candidates because vaccinia is safe and the recombinant virus would most probably elicit not only humoral and CD4 cellular immune responses to HIV but also class I restricted cytotoxic CD8 T-cell responses.10-12 The HIV-1envelope gene has been inserted into the vaccinia virus genome, and cells infected with the recombinant virus express the products of the gpl60 gene on their cell surface, with appropriate post-transcriptional glycosylation and processing of the precursor protein into the HIV-1 envelope glycoproteins gpl20 and gp41.2 Non-human primates inoculated with this recombinant virus vaccine have exhibited antibody and T-cell responses HIV.13,14 Here we describe the safety and to immunogenicity of this live recombinant vaccinia virus vaccine in healthy male volunteers.

Materials and methods

Study population 88 healthy homosexual or bisexual males aged 21-45 years were interviewed for the study; 35 were enrolled. All subjects had to be in good health, have no history of immunosuppressive illness in themselves, their immediate family, or household members, and have no contraindication to the receipt of vaccinia virus vaccine. Also, at 60, 30, and 3 days before receipt of the first dose of vaccine, all had to be normal on physical examination and routine biochemical and haematological tests, and show no evidence of HI V infection as determined by HIV-1 antibody and antigen assays, cultures for HIV, tests for intact delayed cutaneous hypersensitivity (Merieux Multitest), CD4 lymphocyte cell counts and CD4:CD8 ratios, serum immunoglobulin concentrations, and in-vitro lymphoproliferative responses to an array of mitogens and soluble antigens. Furthermore, all their sexual and household contacts had to be normal on physical examination and laboratory testing and to

ADDRESSES:

Introduction One approach to the development of a vaccine against human immunodeficiency virus (HIV) is the use of modified live virus subunit vaccines, in which an HIV gene is inserted into an existing attenuated live virus vector such as vaccinia virus, poliovirus, or adenovirus. 1-9 Live

Departments of Laboratory Medicine, Microbiology, and Medicine and Regional Primate Center, University of Washington, Seattle; Oncogen Corporation, Seattle, Washington, USA; and Bristol-Myers-Squibb Co, Wallingford, Connecticutt, USA (E. L. Cooney, MD, A. C. Collier, MD, P. D. Greenberg, MD, R. W. Coombs, MD, J Zarling, PhD, D. E. Arditti, MN, M. C. Hoffman, BA, S-L. Hu, PhD, Prof L. Corey, MD). Correspondence to Prof L. Corey, Pacific Medical Center ZB-30, 1200 12th Avenue South, Room 9307, Seattle, Washington 98144, USA.

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have no serological evidence of HIV infection, normal CD4 counts, and normal CD4:CD8 ratios. Informed consent for the above tests was obtained from all subjects and household and sexual contacts.

4 replicate antigen-stimulated wells by the mean count of 4 replicate unstimulated control wells. The concentration of antigen or mitogen giving the highest stimulation index was used for these data

analyses. Vaccines

Participants were randomly allocated to receive either standard New York City Board of Health strain vaccinia virus, smallpox vaccine Laboratories, Philadelphia, (’Dryvax’, Wyeth Pennsylvania), or the study vaccine (HIVAC-le), which is a recombinant vaccinia virus expressing the gpl60 envelope gene of HIV (Oncogen, Seattle, Washington). How the vaccine was constructed and produced has been described elsewhereOne lot of vaccine (titre 2 x 109 pfu/ml, supplied as sterile frozen single-dose suspension, was used for all recipients. Participants were stratified by history, physical findings, presence or absence of neutralising antibodies to vaccinia virus, and, by in-vitro lymphoproliferative responses to inactivated vaccinia antigens, as vaccinia primed (vac-p) or vaccinia naive (vac-n, negative in all 4 parameters). The trial was designed as a dose-escalation study. Vac-p individuals were given a 107, 10, or 109 pfu/ml concentrate of dryvax or HIVAC-le at day 0. The 107 and 10 pfu/ml recipients were boosted at day 56 with a dose 1 log higher in concentration than that given on day 0, and those given 109 pfu/ml were given a second dose of 109 pfu/ml. Vac-n subjects recieved 106 pfu/ml of either vaccine at day 0 and a 107 pfu/ml dose at day 56. All vaccines were given by standard intradermal scarification in the deltoid area with 15 pricks of a bifurcated needle for vac-p subjects and 5-8 for vac-n individuals.

Clinical evaluation and follow-up The vaccine site was covered with a 1 x 1 cm gauze and overlaid a 5 x 8 cm transparent dressing (’OpSite’, Smith & Nephew Corp, Massilon, Ohio), which was removed only by the study team. The first 10 recipients of the recombinant vaccine were housed in a closed ward from time of vaccination until healing of the vaccination site and were seen daily. Subsequent recipients of recombinant vaccine, who were followed up as outpatients, and all vaccinia recipients were seen every other day for the first 2 weeks and then every 3-4 days until healing of the vaccination site. At each post-vaccination visit a standardised questionnaire was administered, and the vaccination site was examined, measured, photographed, and cultured for vaccine virus. In selected individuals swabs of the outer surface of the opsite dressing were taken for culture before its removal. Vaccination lesions were classified by WHO criteria as a primary vaccination reaction or a revaccination reaction." Contacts were seen before and 6 weeks after the vaccination. with

Laboratory methods In-vitro T-cell proliferative assays were done on all individuals 60 days before vaccination, on the day of vaccination, and 4, 8, 12, and 16 weeks and 7 months after vaccination. HIV antigens included: ultraviolet inactivated (LJV) and psoralen-inactivated (PI) HIV-1(LAV-BRU strain) at 5, 2-5, 1, and 0-5 ug/ml, live HIV-1 LAV (BRU) (105 TCID), and baculovirus-derived gp160 protein (IIIB strain) at 5 ug/ml (courtesy of MicroGeneSys Inc, West Haven, Connecticutt). The gpl60 protein preparation became available only 12-16 weeks after enrolment. Non-HIV-1 soluble antigens and mitogens included gradient purified UV-inactivated vaccinia virus, heat-inactivated herpes simplex virus and cytomegalovirus, tetanus toxoid (Wyeth Laboratories), dialysed candida antigen (Hollister Stier), concanavalin A (conA), pokeweed mitogen, and phytohaemaglutinin (PHA). Peripheral blood mononuclear cells (PBMC) were obtained by ’FicollHypaque’ separation and stimulated in vitro with mitogens and soluble antigens in 96-well plates for 3 and 6 days, respectively. Each well was then labelled with 2-5 pCi 3H-TdR, the cells were collected by cell-harvester, and 3H-TdR incorporation (A cpm) was calculated as the difference between mean counts (on liquid scintillation counter) of 4 replicate antigen-stimulated wells and the mean counts of 4 replicate unstimulated control wells. The stimulation index (SI) was calculated by dividing the mean count of

Antibodies to HIV-1were assayed before vaccination and every 2 weeks for the first 16 weeks after vaccination by both enzyme immunoassay (EIA) (Genetic Systems, Seattle) and western blot (Epitope Inc, Beaverton, Oregon). All persons with antibodies to HIV envelope detectable by western blot or EIA were tested for HIV-1 neutralising antibodies with the LAV strain of HIV-1 grown in CEM cells. Vaccinia neutralising antibodies were assayed by plaque reduction. The vaccinia neutralising antibody titre was the highest dilution of sera which inhibited 50% of the plaques. Titres of 1/8 were considered negative and all serum samples from an individual were assayed in the same run. Viral isolation for recombinant and standard vaccinia virus was done under P3 containment conditions. Swabs of scarification sites were placed into viral transport medium and inoculated into tube cultures of BSC-40 cells which were examined daily for cytopathic effect (CPE). The titre of virus in cultures of swabs from the lesion was assayed by making serial 10-fold dilutions of transport media and inoculating 0’ 1 ml of the dilutions into duplicate tube cultures. The lesion titre was the highest dilution that produced CPE as calculated with the Reed Muensch formula. Restriction endonuclease analysis was used to evaluate the genetic stability of the recombinant virus after short-term passage in man. DNA from virus isolated from lesion swabs was purified by phenol/chloroform extraction and digested by the restriction16 endonucleases Hind III, NcoI, Scal, PstI, Xho I, and Asp718.16 Digest fragments were resolved by electrophoresis on a 1 % agarose gel and then stained with ethidium bromide. Fragments were also transferred onto a nitrocellulose filter by Southern blot and those containing HIV-specific sequences were detected by hybridisation with a 32P-labelled plasmid DNA pRS3, which contains the entire env region of HIV-1 (BRLI].1’

Statistical methods

Groups were compared by x.2, Fisher’s exact test, or Student’s t For evaluating the in-vitro proliferative responses to UV and PI HIV antigen a positive response was defmed as an SI > 30 in conjunction with A cpm of > 500. For other soluble antigens and mitogens, a positive response was defined as a SI > 3-0. test.

Results

Study population Of the 35 men who were enrolled (all white, mean age 362 31 were vac-p and 4 were vac-n; 28 of 31 vac-p individuals had not received smallpox vaccination within the previous 5 years, and 75% had not been vaccinated within 15 years of enrolment. 15 of the vac-p individuals were vaccinated with vaccinia; 16 received the vaccinia recombinant. 2 of the 4 vac-n subjects received vaccinia and 2 the vaccinia recombinant. The two vaccination groups were similar in age, race, socioeconomic status, media time since last vaccinia vaccination, past number of sexual partners, and duration of homosexual activity.

years),

Clinical and virological responses to vaccination Initial vaccination-Initial vaccination resulted in a clinical "take" in 23 (3 primary, 20 re-vaccination responses) of the 31 vac-p individuals-1of 16 vaccinia recipients and 12 of 15 vaccinia recombinant recipients (table I). Virus was isolated from the lesion site in 3 of 5 given 107 pfu/ml, 4 of 5 given 108 pfu/ml, and all 6 given 109 pfujml vaccinia Details of the techniques briefly described above are available from the The Lancet on request.

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TABLE I-DURATION AND TITRE OF VACCINE VIRUS ISOLATED FROM THE VACCINATION SITE

times

higher among vac-n (107 TCID5o) than among vac-p (105 TCID5o) subjects who received the recombinant vaccine. Booster vaccination-34 of the 35 study participants underwent booster vaccination. Of the 8 individuals who did shed virus after their initial vaccination, 7 had a clinical and virological take after repeat vaccination. Overall, 30 of the 31 vac-p and 34 of 35 individuals who received at least a 107 pfu dose of either vaccine had a clinical and virological take after initial or booster dose. After the booster the duration of viral shedding from the site of the lesion fell (2-0 days, compared with 5-7 days after initial dose, p < 0-01) (table i), as did the mean peak titres of virus (by a hundredfold). None of the vac-n individuals shed virus after booster vaccination.

not

Local reactions and toxicity after vaccination

I

I

I

I

for comparison between first and second doses within vaccinia and vaCCInia recombinant virus recipients Numbers in parentheses refer to SD

*p 14 days after vaccination in 3 of the 4 vac-n individuals, compared with 3 of 31 vac-p (p < 0-01, Fisher’s exact test) subjects. The mean peak titre of virus isolated from vaccination sites was 100

Reactions to vaccination were mild, were reported more frequently with initial than with booster vaccination, and were similar between vaccinia and vaccinia recombinant virus recipients. After the initial dose, pruritus and local soreness at the vaccination site were reported by 16 (94%) and 9 (53%), respectively, of the 17 recombinant virus recipients, compared with 14 (78%) and 6 (33%) of the 18 vaccinia recipients (NS). The mean duration of local itching was 42 for recombinant virus recipients and 5-3 days for vaccinia recipients (NS). Axillary tenderness and fever (> 38 5°C) developed in 3 and 2 individuals, respectively, after vaccination, in all instances after the first dose. No satellite lesions, contiguous spread of virus to other cutaneous sites, or neurological reactions occurred in any of the vaccinees. No subject missed work because of complications of vaccination, and there were no changes in haematological, hepatic, or renal function.

Dressing site cultures and contacts of vaccinees 159 swabs of the opsite dressing from 18 vaccinia recombinant recipients and 31 from 5 vaccinia recipients were sent for culture. 70 swabs were obtained on days when recombinant virus was isolated from the skin site under the dressing and 30 on days when vaccinia virus was isolated. All

Fig 1-Restriction endonuclease (panels 1, 3, 5) and Southern blot (panels 2,4,6) analyses of DNA obtained from viruses isolated from vaccinia gp160 recombinant recipients. Virus was recovered from two recombinant virus recipients (VE0014and VE0041 ) on days 1 and 5 (lanes A) and days 2 and 5 (lanes B) after inoculation. Identical sequences were observed in all viral isolates as well as in the inoculum virus (HIVAC-1 e), but not in the parental vaccinia virus (v-NY).

570

TABLE II-VACCINIA NEUTRALIS ING ANTIBODY AND LYMPHOPROLIFERATIVE RESPONSES

yyt::t::1B

Fig 2-Comparison of in-vitro T-cell proliferative responses to the non-HIV antigens CMV and candida and mitogen PHA in subjects pre (week 0) and post (weeks 4 and 12) vaccination with vaccinia (A) or the vaccinia gp160 recombinant (B). Results are expressed as the mean peak A cpm of the 17 vaccinia and 18 vaccinia recombinant recipients assayed at each time point. Mean response to each antigen or mitogen did not vary significantly over the course of the study m either vaccine group. Similar patterns of response were obtained for other antigens, such as herpes simplex virus and concanavalin A.

cultures of swabs taken of the outer surface of the dressing negative for virus. None of the household or sexual contacts showed rises in their serum vaccinia neutralising antibody titres or cutaneous manifestations consistent with vaccinia exposure.

I

I

*p

75 whenever responses

were

measured.

Lymphoproliferative responses to PHA, conA, pokeweed mitogen, and the soluble antigens candida, tetanus toxoid, cytomegalovirus, and herpes simplex virus did not vary significantly between vaccinia recombinant and vaccinia recipients over the course of the study. Representative data are shown in figure 2. T-cell proliferative responses to HIV antigens Both vac-n recombinant virus recipients developed a T-cell response to all three HIV antigen preparations after vaccination (table III). The mean SI to UV and PI HIV was 71 at week 16; in both subjects the response to this antigen was still present at 12 months’ follow-up. 11 (69%) of the 16 vac-p recombinant virus recipients developed a response to HIV, 6 to one and 5 to at least two of the HIV-antigen preparations. 8 vac-p individuals responded to UV and PI HIV, 5 after the first (mean SI 8-5) and 3 after the second (mean SI 12-6) dose of recombinant vaccine. 4 of these 8 individuals also responded to live HIV and 2 to recombinant gpl60 protein. 2 other vac-p subjects responded only to live HIV and 1 individual responded only to recombinant gpl60. Persisting responses were detected in 2 of 14 vac-p recipients 6-12 months after vaccination. At 12-15 months, 5 of the vac-p subjects were evaluated for a response to nonglycosylated gpl20 envelope protein produced in yeast and derived from the IIIB strain of HIV (kindly provided by K. Steimer, Chiron Corp, Emeryville, California); 4 of these responded (mean SI = 23), and 2 of these 4 had not previously responded to any of the other three HIV antigens. Overall, 13 of 16 (81%) vac-p subjects showed evidence of T-cell priming to HIV by the recombinant virus and

571

TABLE III-LYMPHOPROLIFERATIVE RESPONSES

(SI) TO

HIV ANTIGENS

*SEM NT= Not tested.

(42%) had persisting responses at long-term follow-up. Lymphoproliferative responses to density gradient purified UV and PI ARV, and to HIV-1strain antigenically distinct in the envelope region from LAV, were also assessed in 1 vac-n and 1 vac-p recombinant virus recipient 12-16 weeks after initial vaccination. The SIto UV PI LAV, UV PI ARV, and live LAV were respectively 10-6,54, and 32-6 in the vac-n subject and 4-3, 5-4, and 8-1 in the vac-p individual, indicating that the T-cell response elicited by vaccination includes recognition of shared epitopes. 6

Antibody responses to HIV Antibody responses to HIV envelope proteins developed in 3 of 18 vaccinia recombinant recipients. Both vac-n recombinant virus recipients developed antibody to gpl60, as well as to gp 120 and gp41 by western blot analysis. Antibodies were first detected at week 7 after vaccination and persisted for 7 and 12 months in these 2 individuals (fig 3). Antibodies to HIV developed in only 1 of the 16 vac-p recombinant virus recipients (p 0-02, Fisher’s exact test). =

Weeks Post

Receipt of Vaccinia/Envelope Vaccine

Fig 3-Antibodies to HIV envelope proteins by western blot analysis after vaccination with the vaccinia gp160 recombinant. HIV-1 antigen (whole virus cell lysate Epitope Inc, Beaverton, Oregon) in exposed to a 1 /25 dilution of serum at each time point. Reactivity at 19 000 and 16 000 molecular weight were detected on other recombinant virus recipient blots and probably represent antibodies to HIV envelope products. In this expenment, the non-specific non-viral protein band at approximately 35 000 molecular weight was present before vaccination and was not detected when 2 different lots of viral lysate were used in the assays. All serum samples in this experiment were run in parallel with a

was

single antigen preparation.

This individual shed recombinant virus for 15 days after viral inoculation. Antibodies to gp 160 were first detected by western blot assay at week 12. No vaccinee developed either a positive EIA or neutralising antibodies to HIV after vaccination with the recombinant virus.

Discussion This trial provides several important insights into the part that recombinant vaccinia viruses play in HIV vaccine development and as immunising agents in general. Vaccination with a live recombinant vaccinia virus expressing foreign DNA can be accomplished safely and is well tolerated. After recombinant virus inoculation, clinical reactions were mild and similar to those occurring after administration of standard vaccinia virus. The use of a gauze and non-permeable opsite dressing to cover the vaccination site effectively contained replicating virus, thereby preventing environmental contamination and person-toperson spread of vaccine virus. Additionally, the vaccine virus seems to be genetically stable in vivo, as indicated by restriction endonuclease analysis of sequential viral isolates obtained from recombinant vaccine recipients. In both vac-p and vac-n individuals, the clinical, virological, and immunological responses elicited by inoculation with the vaccinia recombinant seem similar to the responses to vaccination with standard vaccinia virus. In addition, this gpl60 vaccinia recombinant was not associated with either clinical or laboratory evidence of immunosuppression, as shown by stable T-cell responses to an array of non-HIV immunogens over time. Although most of our vac-p subjects had not received smallpox immunisation for over 15 years, all had immunity to vaccinia at study entry as indicated by the presence of serum neutralising antibodies (94%) and/or an in-vitro T-cell proliferative response to vaccinia (81 %). Employing an initial dose of 108 or 109 pfu/ml of virus elicited a clinical take and enhanced T-cell and antibody responses to vaccinia in 30 of 31 vac-p subjects enrolled. However, the responses to vaccinia were much lower after the booster dose than after the first dose of vaccine, even in vac-n individuals. Boosting at week 8 did not elicit a second take or enhance serological or T-cell responses to vaccinia or HIV in any of our vac-n recipients. This finding contrasts with the enhanced vaccinia and HIV responses occurring in macaques and chimpanzees after priming and boosting at week 8 with this vaccine .13,14 These data suggest that long-lasting immunity to vaccinia limits how vaccinia recombinants can be used for repetitive boosting, and that after priming with a novel

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antigen presented as a vaccinia recombinant, other types of vaccines such as recombinant proteins or other modified live viruses should be considered for augmenting host responses. The most important part of our trial was the finding that

6. Putney SD, Matthews TJ, Robey WG, et al. HTLV-III/LAVneutralizing antibodies to an E coli-produced fragment of the virus envelope. Science 1986; 234: 1392-95. 7. Evans DJ, McKeating J, Meredith JM, et al. An engineered poliovirus chimaera elicits broadly reactive HIV-1 neutralizing antibodies. Nature

this recombinant vaccine can elicit both humoral and cellular immune responses to HIV in both vac-p and vac-n individuals. After vaccination, 15/18 (83%) recombinant virus recipients demonstrated T-cell priming to HIV by the development of an in-vitro proliferative response to one or more of the four HIV antigen preparations employed in our assays. Vac-n individuals developed strong and persistent in-vitro T-cell proliferative responses to all HIV preparations used, the T-cell responses remained detectable a year after initial vaccination, and their responses were directed at both the LAV and ARV strains of HIV, which differ signficantly in the envelope regions of their genomes.18 Furthermore, in both vac-n individuals antibodies to HIV envelope proteins developed. The T-cell responses to HIV elicited in vac-p subjects were more restricted and transient than those in vac-n subjects, and in only 1 of 16 did antibodies to HIV envelope develop. This subject was one of 2 vac-p recombinant vaccine recipients who shed recombinant virus from the vaccine site > 14 days after virus inoculation, which suggests that the degree of immunological response elicited by vaccinia recombinants is related to the titre and duration of local virus replication. These observations suggest that the vaccinia gpl60 recombinant primes T and B cells to HIV less effectively in vac-p hosts than in vac-n subjects, and they are in keeping with recently published data on use of vaccinia recombinants expressing HSV gene products in mice.19,2O Our results are in some disagreement with those of Zagury et al, who showed in 1 vaccinia-primed individual given a similarly constructed vaccine both in-vitro lymphoproliferative responses to HIV and neutralising antibody activity to the IIIB strain of HIV.21 Neutralising antibody activity, however, was detected in this individual in the absence of antibodies to HIV envelope of western blot or by whole virus lysate enzyme immunoassay, an unusual

1989; 339:385-88. 8. Dewar RL, Naturajan V, Vasudevachari MB, Salzman NP. Synthesis and processing of human immunodeficiency virus type 1 envelope

finding.22-24 In summary, vaccination with a recombinant vaccinia virus expressing the gp 160 envelope gene of HIV in healthy immunologically competent individuals is safe. Vaccinianaive persons were clearly primed to HIV, as indicated by the development of readily detectable and persistent in-vitro T-cell proliferative and serum antibody responses to HIV. Individuals previously immunised with vaccinia show long-lasting immunity to vaccinia and their priming to HIV by vaccination with this recombinant virus is variable. More recent studies, however, indicate these responses can be significantly augmented by boosting with soluble recombinant envelope proteins. Vaccinia recombinants expressing HIV gene products offer promise in the overall strategy to develop a safe and effective HIV vaccine. REFERENCES Koff WC, Hoth DF. Development and testing of AIDS vaccines. Science 1988; 241: 426-32. 2. Matthews TJ, Bolognesi DP. AIDS vaccines. Sci Am 1988; Oct: 120-27. 3. Hu S-L, Kosowski SG, Dallrymple JM. Expression of AIDS virus envelope gene in recombinant vaccinia viruses. Nature 1986; 320: 1.

537-40.

S, Robert-Guroff M, Wong-Staal F, Gallo RC, Moss B. Expression of the HTLV-III envelope gene by a recombinant vaccinia

4. Chakrabarti

virus. Nature 1986; 320: 535-37. 5. Berman PW, Groopman JE, Gregory T, et al. Human immunodeficiency virus type 1 challenge of chimpanzees immunized with recombinant envelope glycoprotein gp120. Proc Natl Acad Sci 1988; 85:5200-04.

proteins encoded by a recombinant human adenovirus. J Virol 1989; 63: 129-36.

Hung PP, Morin JE, Lubec MD, Expression of HBV surface antigen or HIV envelope protein using recombinant adenovirus vectors. Nat Immun Cell Growth Regul 1988; 7: 135-43. 10. Zagury D, Bernard J, Cheynier R, et al. A group specific anamnestic immune reaction against HIV-1 induced by a candidate vaccine against 9.

AIDS. Nature 1988; 332: 728-31. 11. Takahashi H, Merli S, Putney SD, et al. A single amino acid interchange yields reciprocal CTL specificites for HIV-1 gp160. Science 1989; 246: 118-21. 12. Earl PL, Moss B, Morrison RP, Wehrly K, Nishio J, Chesebro B. T-lymphocyte priming and protection against friend leukemia by vaccinia-retrovirus env gene recombinant. Science 1986; 234: 728-31. 13. Zarling JM, Morton W, Moran PA, McClure J, Kosowski SG, Hu S-L. T-cell responses to human AIDS virus in macaques immunized with recombinant vaccinia viruses. Nature 1986; 323: 344-46. 14. Zarling JM, Eichberg JW, Moran PA, McClure J, Sridhar P, Hu S-L. Proliferative and cytotoxic T cells to AIDS virus glycoproteins in chimpanzees immunized with a recombinant vaccinia virus expressing AIDS virus envelope glycoproteins. J Immunol 1987; 139: 988-90. 15. WHO Expert Committee on Smallpox Eradication, 2nd report. WHO Tech Rep 1972; 493: 36. 16. Joklik WK. The purification of four strains of poxvirus. Virology 1962; 18: 9-18. 17. Wain-Hobson S, Sonigo P, Danus O, Alizon M. Nucleotide sequence of the AIDS virus, LAV. Cell 1985; 40: 9-17. 18. Myers G, Josephs SF, Rabson AB, Smith TF, Wong-Staal F. (eds) Human retroviruses and AIDS. Los Alamos: Los Alamos National Laboratory, 1989. 19. Rooney JF, Wohlenberg C, Cremer KJ, Moss B, Notkins AL. Immunization with a vaccinia virus recombinant expressing herpes simplex virus type 1 glycoprotein D: long-term protection and effect of revaccination. J Virol 1988; 62: 1530-34. 20. Flexner C, Murphy BR, Rooney JF, et al. Successful vaccination with a polyvalent live vector despite existing immunity to an expressed antigen. Nature 1988; 335: 259-62. 21. Zagury D, Leonard R, Fouchard M, et al. Immunization against AIDSin humans. Nature 1987; 326: 249-50. 22. Guroff RM, Brown M, Gallo RC. HTLV-III neutralizing antibodies in patients with AIDS and AIDS related complex. Nature 1985; 316: 72. 23. Lane HC. Evaluation of a recombinant HIV-1 envelope protein as an immunogen in humans. Abstract WCO 20, V International Conference on AIDS, Montreal, Canada, June 4-9, 1989. 24. Dolin R, Graham B, Greenberg S, et al. Safety and immunogenicity of HIV-1 recombinant gp160 vaccine candidate in normal volunteers. Abstract CO 33, V International Conference on AIDS, Montreal,

Canada, June 4-9, 1989.

From The Lancet The Founder I have recently had the curiosity to go through the files of The Lancet with the object of discovering and recording if possible the tone and aims of that journal from its inception, and the enormous labour has been amply repaid by the discovery of the rich mine of wealth which the volumes contain. I believe I have discovered the secret of the unparalleled success and the high reputation of the chief organ of the medical profession. In one of the volumes I came across what may be called The Lancet’s confession of faith. "The Lancet will perpetuate the memory of Thomas Wakley-the founder of this journal-by cherishing after his death the principles to which he consecrated his life." What had he to gain in requiring that medical men should be adequately rewarded for their labours? "I have," said Mr Wakley in a public speech-"I have foresworn medical practice. I use only the lancet, and that in the form of a quill." From that time he became the champion of medical reform of all kinds. ...

Safety of and immunological response to a recombinant vaccinia virus vaccine expressing HIV envelope glycoprotein.

In a randomised phase I trial of a recombinant vaccina virus vaccine expressing the gp160 envelope gene of the human immunodeficiency virus (HIVAC-1e)...
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