Vaccine 29 (2011) 83098316

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Vaccine
journal homepage: www.elsevier.com/locate/vaccine

Safety and immunogenicity of a modied pox vector-based HIV/AIDS vaccine candidate expressing Env, Gag, Pol and Nef proteins of HIV-1 subtype B (MVA-B) in healthy HIV-1-uninfected volunteers: A phase I clinical trial (RISVAC02)
Felipe Garca a,,1 , Juan Carlos Lpez Bernaldo de Quirs b,1 , Carmen E. Gmez c,1 , Beatriz Perdiguero c,1 , Jose L. Njera c,1 , Victoria Jimnez c,1 , Juan Garca-Arriaza c,1 , Alberto C. Guardo a,1 , Inaki Prez a,1 , a,1 b,1 d,1 Vicens Daz-Brito , Matilde Snchez Conde , Nuria Gonzlez , Amparo Alvarez d,1 , Jos Alcam d,1 , Jos Luis Jimnez b,1 , Judit Pich a,1 , Joan Albert Arnaiz a,1 , Mara J. Maleno a,1 , Agathe Len a,1 , Mara Angeles Munoz-Fernndez b,1 , Peter Liljestrm e,1 , Jonathan Weber f,1 , Giuseppe Pantaleo g,1 , Jos M. Gatell a,1 , Montserrat Plana a,1,2 , Mariano Esteban c,1,2
a

Hospital ClinicIDIBAPS, Barcelona, Spain Hospital Gregorio Maran, Madrid, Spain n c Centro Nacional de Biotecnologa, CSIC, Madrid, Spain d AIDS Immunopathogenesis Unit, Centro Nacional de Microbiologa, Instituto de Salud Carlos III, Madrid, Spain e Karolinska Institutet, Stockholm, Sweden f Department of Medicine, Imperial College, London, UK g Division of Immunology and Allergy, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Switzerland
b

a r t i c l e

i n f o

a b s t r a c t
Background: To investigate the safety and immunogenicity of a modied vaccinia virus Ankara vector expressing HIV-1 antigens from clade B (MVA-B), a phase-I, doubled-blind placebo-controlled trial was performed. Methods: 30 HIV-uninfected volunteers at low risk of HIV-1 infection were randomly allocated to receive 3 intramuscular injections (1 108 pfu/dose) of MVA-B (n = 24) or placebo (n = 6) at weeks 0, 4 and 16. All volunteers were followed 48 weeks. Primary end-points were adverse events and immunogenicity. Results: A total of 169 adverse events were reported, 164 of grade 12, and 5 of grade 3 (none related to vaccination). Overall 75% of the volunteers showed positive ELISPOT responses at any time point. The magnitude (median) of the total responses induced was 288 SFC/106 PBMC at week 18. Antibody responses against Env were observed in 95% and 72% of vaccinees at week 18 and 48, respectively. HIV-1 neutralizing antibodies were detected in 33% of volunteers. Conclusions: MVA-B was safe, well tolerated and elicited strong and durable T-cell and antibody responses in 75% and 95% of volunteers, respectively. These data support further exploration of MVA-B as an HIV-1 vaccine candidate. Clinical Trials.gov identier: NCT00679497. 2011 Elsevier Ltd. All rights reserved.

Article history: Received 31 March 2011 Received in revised form 29 June 2011 Accepted 19 August 2011 Available online 9 September 2011 Keywords: MVA-B HIV Vaccine

1. Introduction Given the relentless persistence of the HIV epidemic in the face of anti-retroviral therapy, there is an urgent need to develop preventative approaches which will limit HIV transmission. Recently,

Corresponding author at: Infectious Diseases Unit, Hospital Clnic, Villarroel, 170, 08036 Barcelona, Spain. Tel.: +34 932275586; fax: +34 934514438. E-mail address: fgarcia@clinic.ub.es (F. Garca). 1 For the RISVAC-02 Study. 2 Both authors contributed equally. 0264-410X/$ see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2011.08.098

advances in HIV prevention via preexposure prophylaxis [1], topical microbicides [2] and a weakly effective prime-boost vaccine [3] have been reported. Despite these advances, a safe and potent vaccine remains the most powerful tool for epidemic control of infectious diseases. A phase IIb clinical trial (the STEP study) using serotype 5 adenovirus expressing HIV-1 Gag/Pol/Nef failed to demonstrate prevention of HIV-1 infection or reduction of early viral load [4,5]. Conversely, a combination of a recombinant canarypox vector vaccine (ALVAC-HIV [vCP1521]) plus a recombinant HIV-1 glycoprotein 120 (gp120) subunit vaccine (AIDSVAX B/E) had an efcacy of 31.2% [3]. While this last trial showed only a modest benet, the results offer insight for future research and have

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renewed interest in the development of poxviruses as vectors capable of stimulating the cellular and humoral arms of the immune response [6]. The best studied vaccine vectors in humans are the pox viruses. Particular attention has been, however, focused on pox viruses with limited in vivo replicative capacity and, therefore, non pathogenic in animal models and humans [7,8]. The modied vaccinia virus Ankara (MVA) strain has deletions of the genes associated with pathogenicity. During extensive eld studies, including high risk patients, no side effects were associated with the use of the MVA vaccine [7,9]. MVA vectors expressing different HIV-1 genes have been tested in phase I clinical trials in humans. These have appeared to be safe and well tolerated [1012]. However, limited immunogenicity has been usually reported when MVA is used alone [1116]. This low level of response is similar to other poxvirus used alone [1719]. On the other hand, recent phase I/II clinical trials using either MVA [20] or NYVAC (derived from the Copenhagen strain of vaccinia virus) [21] have shown that poxvirus-based HIV-1 vaccine candidates are highly immunogenic in primeboost immunization regimens with DNA. We report here the results of the rst-in-man phase-I, doubled blind placebo-controlled trial in healthy volunteers that was performed to investigate the safety and immunogenicity of an HIV/AIDS vaccine candidate referred as MVA-B. Previous studies in human dendritic cells infected with MVA-B revealed that the inserted HIV-1 genes induced the expression of cytokines, cytokine receptors, chemokines, chemokine receptors and molecules involved in antigen uptake and processing, including the major histocompatibility complex genes, that might act as regulators of immune responses to HIV-1 antigens [22]. In preclinical studies, MVA-B induced robust HIV specic immune responses in mice that are broad, polyfunctional and durable [23,24] and a similar MVA construct but expressing the HIV-1 89.6p (gp120) and the polyprotein GagPolNef of SIVmac239 induced in macaques specic HIV/SIV immune responses and trigger high and long-term protection following challenge with pathogenic SHIV89.6p [25].

reactions including induration); grade 3 or above systemic adverse event (temperature, chills, headache, nausea, vomiting, malaise, and myalgia); grade 3 or above other clinical or laboratory adverse event conrmed at examination or on repeat testing respectively; any event attributable to vaccine leading to discontinuation of the immunization regimen. Data on local and systemic events listed above were solicited with specic questions for a minimum of 7 days following each immunization. Data on other clinical and laboratory events were collected with an open question at each visit and through routine scheduled investigations respectively. The severity of each adverse event was graded by the investigator. Criteria for grading clinical and laboratory events were based on systems in use at the MRC CTU, Aventis Pasteur and NIH Division of AIDS. The investigator stated the relationship of each adverse event to the study medication using one of the terms dened as unrelated, unlikely to be, possibly, probably and denitely related to vaccination. The primary immunogenicity parameters were quantitative or present/absent, and were cellular responses CD8/CD4+ T-cell responses (ELISPOT) at week 6/8, 18/20, and at any point following both immunizations. Secondary endpoints were safety and immunogenicity as dened as follow: grade 1 and 2 adverse events within 28 days of a vaccination, antibody responses (ELISA for antibodies reactive against HIV-1 Env and the vaccinia virus vector), and neutralizing antibody titers against vaccinia virus and HIV-1 clade B isolates. Study weeks were at week 6 (screening), 0 (rst immunization), 0 + 3 days, 1, 4 (second immunization), 4 + 3 days, 5, 6, 8, 16 (third immunization), 16 + 3 days, 17, 18, 20 and 48. Physical examination, clinical symptoms and adverse events were assessed at each visit. Laboratory hematology and biochemistry were assessed at weeks 6, 0 + 3 days, 4, 4 + 3 days, 8, 16, 16 + 3 days, 20 and 48. A pregnancy test was performed at weeks 0, 4 and 16. Safe sex counseling was performed at weeks 6, 4, 16 and 48. CD4+ T cell count were assessed at weeks 6 and 16. HIV test was performed at screening visit and at weeks 4, 16 and 48 (Fig. 1B).

2. Subjects and methods 2.1. Subjects and samples This was a double-blind randomized phase I trial compared with placebo. A total of 30 HIV-uninfected male and female volunteers at low risk of HIV-1 infection, and with absence of smallpox specic antibodies and no history of previous smallpox vaccination were included in 2 Spanish Clinical Centers in Madrid and Barcelona. Some samples were gently processed by the Spanish HIV BioBank [26]. The study was explained to all subjects in detail, and all gave a written informed consent and were randomly allocated (with balanced randomization [1:1] through a computer generated randomization list without any restriction) to receive 3 intramuscular injections (1 108 pfu/dose) of MVA-B (n = 24) or placebo (n = 6) at weeks 0, 4 and 16. MVA-B was generated as described [23], and the GMP lot was provided by the EuroVacc Foundation. All volunteers were followed until week 48. The study was approved by the institutional ethical review board and by the Spanish Regulatory Authorities. Inclusion criteria were age between 18 and 55 years, at low risk of HIV-1 infection and that accepted to use an effective method of contraception with partner from 14 days prior to the rst vaccination until 4 months after the last immunization. The primary end-points were grade 34 adverse events (AE), and immunogenicity (T-cell responses at week 6, 8, 18 and 20 and at any point following immunizations). The primary safety parameters were graded as: grade 3 or above local adverse event (pain, cutaneous

2.2. Immunogenicity evaluations 2.2.1. ELISPOT assays The immunogenicity of MVA-B was assessed on cryo-preserved blood mononuclear cells at weeks 0, 4, 6, 8, 16, 18, 20 and 48 by the quantication of T-cell responses evaluated by a validated IFNELISPOT assay according to standardized operating procedures in a single research laboratory. ELISPOT assays were performed following the manufacturers instructions (BD Biosciences). Briey, cryo-preserved blood mononuclear cells (PBMCs) were thawed and rested for 8 h at 37 C, and then 200,000 cells were stimulated with peptide pools (1 g of each single peptide) in 100 l of complete media (RPMI plus 10% FCS) in quadruplicate conditions. All peptides used in this study (provided by the EUROVACC Foundation and JPT Peptide Technologies, Germany) were HPLC puried (>80% purity). We used 8 pools of 5061 overlapping peptides each (15 mers with 11 overlap) encompassing the HIV-1 GagPolNef, and Env regions from clade B based on the sequence included in the virus vector. Media only was used as negative control. PHA-P (1 g/ml) and stimulation with CEF were used as positive controls. Results are expressed as the mean number of spot forming cells (SFC)/106 cells from quadruplicate assays. The following criteria were used to dene the technical validity and positive responses: cell samples viability should be >80% to be analyzed; the assay background (media only) had to be <50 SFC/106 PBMC; positive responses against PHA-P had to be above 500 SFC/106 PBMC; and positive ELISPOT responses were considered when they were >50 SFC/106 PBMC and at least 3-fold over media control.

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Fig. 1. Disposition of participants (A) and ow chart of the study (B). A total of 356 subjects were screened for participation in this study. 30 were randomized (24 to MVA-B arm and 6 to placebo arm) and none of them were excluded. All the volunteers received 3 vaccines, 28 completed the study as per protocol and 2 were lost to follow up before week 48 (one at week 18 and another at week 20). The 30 participants were included in the safety analysis.

2.2.2. Antibody responses Antibodies to Env and vaccinia virus proteins in serum were assessed at weeks 0, 8, 18, and 48 using ELISA according to standardized operating procedures in a single research laboratory as previously described [23,24]. To evaluate neutralizing antibody titers against HIV-1 a reporter virus carrying a Renilla luciferase gene in nef was generated by cloning the BX08 full-length envelope in the pNLlacZ/EnvRen vector, as previously described [27]. Viral stocks were generated by transfection in HEK 293 T cells. Titrated recombinant viruses were preincubated with serial 4-fold dilutions of sera (1/41/161/641/256) for 1 h at 37 C before the infection of the U87.CD4.CCR5 cells. Virus infectivity was determined 48 h postinoculation by measuring luciferase activity in cell lysates using a 96-well plate luminometer (Orion, Berthold).

Table 1 Number of side effects reported by volunteers according to relationship with vaccination after each vaccination. Relationship Treatment group Number of side effects Day 0 to week 4 Not related Unlikely Possible Probable Denite MVA HIV-B Placebo MVA HIV-B Placebo MVA HIV-B Placebo MVA HIV-B Placebo MVA HIV-B Placebo 3 2 6 1 20 3 14 36 Week 4 to week 16 8 6 1 25 2 7 1 12 Week 16 to week 48 7 1 6 1 3 4 18 2 13 2 51 6 24 1 52 All

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F. Garca et al. / Vaccine 29 (2011) 83098316 Table 2 Proportion of volunteers suffering local or systemic side effects after any vaccination. Adverse eventa MVA-B 24 (100) 4 (17) 4 (17) 2 (8) 24 (100) 24 (100) 10 (42) 12 (50) 4 (17) 3 (13) 1 (4) 2 (8) 13 (54) 13 (54) 3 (13) Placebo 1 (17) 1 (17) 1 (17) 2 (33) 2 (33) 1 (17) 2 (33) 1 (17) 1 (17) 1 (17) 3 (50) 3 (50)

100

proportion of responders

90 80 70 60 50 40 30 20 10
0

Local reactogenicity, n (%) Pain Redness Itching Induration Any local reaction Grade 12b Grade 34 Systemic reactogenicity, n (%) Headache Malaise Myalgia Nausea/vomiting Chills Fever Any systemic reaction Grade 12c Grade 3d

16
WEEKS

18

20

48

any

proportion of responders

100 90 80 70 60 50 40 30 20 10
0

gag gpn env

a Only adverse events (AE) considered as denitely, probably or possible related to vaccination are included. b Only 4 individuals had grade 2 local reactogenicity. c There were no grade 2 systemic reactions. d There were 5 AE grade 3 in 3 volunteers, but none was considered related to vaccination (1 volunteer tonsillitis, 1 volunteer trafc accident and 1 volunteer had 1 pneumonia and 2 asthmatic attacks).

mine whether there is or not a signicant difference in the levels of antibodies between two weeks, we used Wilcoxon Signed-Rank Test. 3. Results
0 4 6 8 16
WEEKS

18

20

48

any

3.1. Clinical characteristics and safety Healthy volunteers were recruited through advertising in hospitals, colleges, newspapers and magazines and given a telephone number to contact (n = 356). The interested subjects were provided with further information about the study, and asked to complete a short interview (by telephone or in person) to assess their suitability (pre-screening, n = 171). If they were still interested and willing to participate, they were invited to attend for a screening visit (n = 83) and at this point they were allocated a number for the screening register (Fig. 1A). Thirty volunteers were randomized (24 to MVA-B arm and 6 to placebo arm) and none of them were excluded. All the volunteers received 3 vaccines given by intramuscular route, 28 completed the study as per protocol and 2 were lost to follow up before week 48 (one after week 18 and another after week 20). The 30 participants were included in the safety analysis. The median age was 27 years (range 1948). Twenty-four (80%) were male and 27 Spanish (90%). Overall the vaccine was well tolerated. A total of 169 adverse events (AE) were reported during follow-up (158 in vaccinated and 11 in placebo group). One hundred and forty-ve AE were grade 1, 8 grade 2 and 5 grade 3. Fifty-two of grade 12 AE were considered as denitely related to vaccination (4 out of 8 grade 2 AE were considered as denitely related). There were 5 AE grade 3 in 3 volunteers (all in vaccinated group), but none was considered related to vaccination (1 volunteer had tonsillitis, 1 volunteer had a trafc accident and 1 volunteer had 1 pneumonia and 2 asthmatic attacks). The number and relationship of AE after each vaccination are shown in Table 1. The number of AE considered as denitely or probably related to vaccination decreased progressively from the rst to third vaccination. The percentage of volunteers experiencing local and systemic AE is presented in Table 2. The most frequently reported local

Fig. 2. Proportion of responders (volunteers with positive IFN- ELISPOT responses) at each time point. (A) Proportion of responders to MVA-B at the different time points. (B) Percentage of responders to HIV-1 Gag, GPN (GagPolNef polyprotein) and Env regions at the different time points. Positive IFN- ELISPOT responses were considered when they were >50 SFC/106 PBMC and at least 3-fold over media control. Red, blue, green and black bars represent proportion of responders against Gag, GPN (GagPolNef polyprotein), Env and any (total) regions from clade B included in the virus vector, respectively. Arrows represent the different doses of MVA-B vaccine. Any represent proportion of responders at any time point.

2.3. Statistical analysis It is not the remit of this study to recruit a sufcient number of participants to be statistically condent about the result. However by the end of this study at least 24 participants will have been exposed to the study vaccine schedule of interest and this provides the following condence around observed severe event proportions of 0, 1, 2 and 3 as follows: number of events: 0 (95% condence interval, CI: 014%), 1 (95% CI: 0.121%), 2 (95% CI: 0.127%), 3 (95% CI: 2.632%). The safety endpoints were described and summarized by number and percentage of adverse events and grading. All clinical event and routine laboratory data were included in the safety analysis. This is an exploratory study, and the immunological analyses will be descriptive. The T-cell responses were analyzed as present or absent and reported as the number and proportion of participants responding to each peptide pool and for each time point. The total ELISPOT responses were described as the sum of SFC of all positive responses, per peptide pool or after grouping pools from the same HIV-1 protein, after substraction of background. Each participant was classied as a responder if there was at least one positive against any of the HIV-1 peptides pools at any time, and nonresponder if ELISPOT responses were all negative. To deter-

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TOTAL

2000 1000 1000 900

SFC/106 PBMC

800 700 600 500 400 300 200 100 0 0 4 6 8 16 18 20 48

WEEKS

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gpn
1000 500 500 450 400 350 300 250 200 150 100 50 0 1000 500 500 450 400 350 300 250 200 150 100 50 0

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SFC/106 PBMC

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SFC/106 PBMC

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Fig. 3. A. Magnitude of total HIV-1-specic T-cell responses against any HIV-1 region from clade B included in the virus vector. Horizontal line represents the median value, red circles individual values at each time point. Arrows represent the different doses of MVA-B vaccine. Dashed line represents the limit considered as positive response (those >50 SFC/106 PBMC and at least 3-fold over media control). B. Magnitude of HIV-1-specic T-cell responses against HIV-1 Gag, GPN (GagPolNef polyprotein) and Env regions from clade B included in the virus vector. Horizontal line represents the median value, red circles individual values at each time point. Arrows represent the different doses of MVA-B vaccine. Dashed line represents the limit considered as positive response (those >50 SFC/106 PBMC and at least 3-fold over media control).

reactogenicity AE was pain that appeared in all vaccinees, although only in 1 subject was grade 2. The most frequently reported systemic reactogenicity events were headache and malaise, and both of them were grade 1. The median (IQR) duration of local AE was 3days (IQR 23), the systemic AE 1.5 days (IQR 12). There were no laboratory changes considered as grade 3 or 4. 3.2. Cellular immunogenicity Overall 75% of the vaccinated volunteers responded, as measured by ELISPOT, to any pool included in the vector at any time point. Positive T-cell responses were detected by ELISPOT in 58%, 67%, 50% and 68% of vaccinees, at weeks 6, 8, 18 and 20, respectively. The proportion of responders increased clearly after the second dose of vaccine from 33% of responders at week 4 (second dose) to 67% at week 8. At week 16 (third dose) the proportion of responders decreased to 50% to increase again up to 68% at week 20. Vaccine-specic responses were maintained at least until week 48 in 15/22 (68%) individuals (Fig. 2A). The proportion of responders against Gag, GPN (GagPolNef polyprotein), and Env pools at any time point was 38%, 46% and 67%, respectively. Vaccine induced T-cell responses were predominantly directed against Env peptide pools. At weeks 6, 8, 18 and 20 (primary endpoints), Env-specic responses were observed in 50%, 58%, 38% and 50% of vaccinees

whereas Gag and GPN induced T-cell responses were observed in a range of 13% to 21% of individuals (Fig. 2B). Concerning the breadth of the immune response, 7 (29%) immunized volunteers responded exclusively to a unique antigen (6 (25%) to Env and 1 (4%) to GNP), and none to Gag whereas 11 (46%) vaccinated volunteers showed positive responses to at least 2 different antigens (6 (25%) to Env and GPN, 1 (4%) to Env and Gag, and 4 (17%) to Env, GPN and Gag). All these responses were consistently being observed from weeks 46 and at all timepoints until almost the end of the follow-up (weeks 20 or 48) (data not shown). No HIV-1 positive T-cell responses were observed in placebo arm except in one of the participants receiving placebo (#209) that had detectable ELISPOT responses against GPN2 pool at baseline and at all subsequent weeks without changes in the magnitude being probably a false positive (data not shown). Regarding the magnitude of the response, the median of the total responses induced was 236, 139, 288 and 156 SFC/106 PBMC at weeks 6, 8, 18 and 20, respectively and was similar against HIV-1 Gag, GPN and Env pools. The peak of the magnitude of the total response was at week 4 with a median of 474 SFC/106 PBMC (range 193589). At this time, after the rst immunization, the greatest magnitude was against Env pools (median: 209, range: 123399 SFC/106 PBMC). By contrast, we observed that the higher responses against Gag and GPN were observed at week 18, after the third immunization (medians: 239 and 143 SFC/106 PMBC,

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p= 0.06

A
Log Mean Titer

100000 10000 1000 100 10 <50

V

p= 1.43e-05

p= 3.13e-05

B
Frequency of responders (%)
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80 60 40 20 0
45.8% 95.8 % 72.7%

18

48

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10000 1000 Log Mean Titer 100 10 <50

p= 2.24e-04 p= 3.76e-05 p= 2.14e-05

D
Frequency of responders (%) 100 80 60 40 20 0 8 18 48
91.7% 100 % 77.3%

18

48

Weeks

Weeks
Fig. 4. Binding antibody response rate against HIV-1 gp160 (A and B) and anti-vaccinia binding antibodies (C and D) at weeks 8, 18 and 48. A and C. Magnitude. B and D Proportion of responders. Circles represent individual values. Dashed line represents the threshold considered as positive response. V: Vaccinated. P: Placebo.

respectively). At week 48, the magnitude of the total response decreased to a median of 132 SFC/106 PBMC (range 52690) (Fig. 3 and Table 3). 3.3. Humoral immunogenicity Binding antibodies to gp160. As shown in Fig. 4A and B, 11 out of 24 volunteers (45.8%), 23 out of 24 (95.8%), and 16 out of 22 (72.7%) formed binding antibodies against HIV-1 subtype B gp160 at weeks 8, 18 and 48, respectively. None of the placebo recipients formed positive responses. Signicantly, the anti-Env response was enhanced about 2-fold after the third dose of the vaccine (p = 1.43e05 ) decreasing again at week 48 (p = 3.13e05 ). One vaccinated volunteer showed a positive HIV-1 ELISA test but with a

Table 3 Magnitude of T-cell responses, measured at each time point by IFN- ELISPOT, against HIV-1 Gag, GPN (Gag-Pol-Nef polyprotein) and Env peptide pools and any regions from HIV-1 clade B included in the virus vector. Week 0 4 6 8 16 18 20 48 Gag 0 (0) 135 (103496) 116 (56178) 75 (55920) 145 (50404) 239 (119313) 86 (50216) 151 (69216) GPN 0 (0) 95 (71164) 90 (54253) 106 (55524) 90 (83198) 143 (94491) 56 (53178) 114 (63283) Env 0 (0) 209 (123399) 193 (50716) 115 (51896) 125 (76293) 172 (70626) 149 (81558) 111 (54690) Total 0 (0) 474 (193589) 236 (501003) 139 (511680) 198 (99528) 288 (70745) 156 (81608) 132 (55690)

negative Western Blot on week 4, becoming both tests negative on weeks 16 and 52 (data not shown). Anti-vaccinia antibodies. Fig. 4C and D depicts antibody titers against vaccinia virus proteins over time in vaccine and placebo recipients. Anti-vaccinia titers were high after the second immunization. The proportion of responders increased from 91.7% at week 8100% at week 18. Thereafter, it decreased to 77.3% at week 48. These titers are considerably lower than those induced by the smallpox vaccine [28]. Neutralizing antibodies. Neutralizing activity from serum of volunteers was measured at week 48 (in subjects # 102 and #106 was determined at week 18 and 20 respectively) against HIV-1 primary isolate BX08 (Fig. 5). After three vaccinations of MVA-B, 8 out of 24 volunteers (33%) were able to neutralize with a titer >1/90 (that we consider as positive). If we include also patients with a weak positive response (a titer above 1/50) then 13 out of 24 volunteers (54%) show a positive response. None of the placebo recipients gave positive responses. There were no cross-clade neutralizing antibody responses, as no neutralizing activity was detected in serum against virus MN and AC10 (data not shown). 4. Discussion The results of this phase I clinical trial showed that the HIV/AIDS vaccine candidate MVA-B was safe and well tolerated. Although all the volunteers that received the vaccine suffered at least one side effect, a 92% of these side effects were considered as grade 1. There were no grade 3 or 4 side effects related to vaccination and only 4 out of 8 grade 2 side effects were related to vaccination.

Median (range) SFC/106 PBMC.

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Fig. 5. Neutralizing activity of serum against primary viral isolate BX08 at week 48. Results are shown as the inverse of IC50 values. Black triangles show individual neutralization values in placebo recipients and red squares values in vaccinated group at week 48. The median value is shown.

Interestingly, the number of side effects decreased with each dose of vaccination (Table 1). These data support the previous nding that MVA has a good safety prole [7,9]. This HIV/AIDS vaccine elicited strong T-cell responses in 75% of volunteers, that were maintained until week 48 in 68% of individuals. The proportion of responders against HIV-1 Gag, GPN (GagPolNef polyprotein), and Env pools at any time point was 38%, 46% and 67%. This predominant response against Env has been already observed with the poxvirus strain NYVAC [18,19,21]. The proportion of responders increased clearly after the second dose of vaccine from 33% of responders at week 4 (before second dose) to 67% at week 8 (after second dose). At week 16 (before third dose) the proportion of responders decreased to 50% to increase again up to 68% after the third dose of vaccine. Although it seems that a third dose could not increase further the proportion of responders (as comparing numbers of responders at weeks 8 and 20), it could be speculated that 3 doses could be important to maintain the high level of response at mid-term (week 48), given that an increase in the proportion of responders is observed from week 16 (before third vaccination) to week 20 and 48 (after the third dose). Moreover, and concerning the breadth of the response, 46% of the vaccinees developed positive and consistent responses until the end of the follow-up to at least two of the antigens vs. 29% volunteers who showed reactivity against only one HIV antigen (Env > GPN). The median of magnitude of the response measured by ELISPOT was 288 SFC/106 PBMC after the third dose of vaccine and was maintained above 100 SFC/106 PBMC at week 48. Conversely to proportion of responders, the magnitude of response was similar against HIV-1 Gag, GPN and Env pools, although the earlier and more frequent response was against Env peptide pools. It can be speculated that a third boost with MVA-B may be necessary to improve and amplify the T-cell responses to other epitopes such as Gag or GPN, since the magnitude of the response increased after the third dose of vaccine. The results of this phase I clinical trial suggest that MVA-B is immunogenic in most of the individuals tested (75% of responders). Volunteers in the STEP trial received 3 doses of serotype 5 adenovirus expressing HIV-1 Gag/Pol/Nef and elicited IFNELISPOT responses in 75% of vaccines [4,5]. Moreover, the range of responses in the STEP trial was 163686 SFC/106 PBMC, similar to the range of 70745 SFC/106 PBMC observed in our trial after the third boost. Other MVA vectors expressing different HIV genes have shown limited immunogenicity [13], where cell-mediated immune responses were reported to be moderate in response rate (17%) [11,12] and in magnitude (median IFN-gamma ELISPOT responses below 100 SFC/106 PBMC) [11,12,1416]. It seems that higher doses enhanced the response rate (50100%), but the magnitude of the

response remained low [1416]. This low level of response is similar to other poxvirus used alone [17,19]. On the other hand, recent phase I/II clinical trials using either MVA [20] or NYVAC [21] have shown that poxvirus-based HIV vaccine candidates are highly immunogenic in primeboost immunization regimens with DNA. Our data show that MVA-B deserves to be considered as a potential HIV/AIDS vaccine candidate to be used alone and in primeboost combination with other immunogens in future trials. Regarding humoral immunogenicity, the proportion of responders against HIV-1 gp160 was high (95%) and durable. A third booster dose increased by 2-fold the frequency of responders to Env while the proportion of responders to vaccinia antigens was maintained after the second dose and decrease with time. Thus, three doses of MVA-B are necessary to stimulate antibody levels against HIV Env. We also found that MVA-B was able to generate neutralizing activity of serum in at least a third of volunteers. Given the promising results of a combination of a recombinant canarypox vector vaccine (ALVAC-HIV [vCP1521]) plus a recombinant gp120 subunit vaccine (AIDSVAX B/E) [3], our ndings would support using MVA-B in a prime-boost regimen together with a recombinant glycoprotein in adjuvant as a booster to further increase the neutralizing antibody response. 5. Conclusions This rst phase I trial with the HIV/AIDS vaccine candidate MVAB in healthy volunteers showed that this immunogen was safe and well tolerated. In addition, this vaccine elicited strong and durable T-cell responses in 75% of volunteers. In the vaccinees it also elicited 95% of antibody responses against HIV-1 Env and 33% of patients generated neutralizing antibodies. These data support further exploration of MVA-B as an HIV-1 vaccine candidate. Acknowledgements This study was partially supported by grants: FIS PS09/01297, SAF2006-26667-E, SAF2008-02036, FIS PI070291, FIS PI050058, SAF 05/05566, TRA-094, EC10-153, NAN2007-31198-E, PI09/02029, PI061468, FIPSE Fundacin para la investigacin y prevencin del sida en Espana 240800/09, Foundation Marcelino Botn, RIS Red Temtica Cooperativa de Grupos de Investigacin en Sida del Fondo de Investigacin Sanitaria (FIS), HIVACAT HIV development program in Catalonia. Instituto de Salud Carlos III (FIS PI080752, ISCIII-RETIC RD06/0006) the Agence Nationale de Recherches sur le SIDA (ANRS) and FIPSE (36536/05, 36630/07). The research leading to these results has received funding from the European Communitys Sixth and Seventh Framework programmes (FP7/2007-2013) under grant agreement no. 242135 and EUROPRISE Network of Excellence (LSHP CT-2006-037611). Dr. Felipe Garca was recipient of a Research Grant from IDIBAPS Institut dInvestigacions Biomdiques August Pi I Sunyer, Barcelona, Spain. Dr. Montserrat Plana is a researcher from the Institut dInvestigacions Biomdiques August Pi i Sunyer (IDIBAPS) and is supported by the Spanish Health Institute Carlos III (ISCIII) and the Health Department of the Catalan Government (Generalitat de Catalunya). Dr. Jos Luis Jimnez was supported by FIS PI081495 and programa de investigacin de la Consejera Sanidad de la Comunidad de Madrid. This study has been presented in part at 18th Conference on Retroviruses and Opportunistic Infections (CROI) (Boston, February 27 to March 2, 2011). Abstract 373.

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F. Garca et al. / Vaccine 29 (2011) 83098316 [9] Stickl HA. Smallpox vaccination and its consequences: rst experiences with the highly attenuated smallpox vaccine MVA. Prev Med 1974;3:97101. [10] Mwau M, Cebere I, Sutton J, Chikoti P, Winstone N, Wee EG, et al. A human immunodeciency virus 1 (HIV-1) clade A vaccine in clinical trials: stimulation of HIV-specic T-cell responses by DNA and recombinant modied vaccinia virus Ankara (MVA) vaccines in humans. J Gen Virol 2004;85:9119. [11] Cebere I, Dorrell L, McShane H, Simmons A, McCormack S, Schmidt C, et al. Phase I clinical trial safety of DNA- and modied virus Ankara-vectored human immunodeciency virus type 1 (HIV-1) vaccines administered alone and in a prime-boost regime to healthy HIV-1-uninfected volunteers. Vaccine 2006;24:41725. [12] Goonetilleke N, Moore S, Dally L, Winstone N, Cebere I, Mahmoud A, et al. Induction of multifunctional human immunodeciency virus type 1 (HIV-1)-specic T cells capable of proliferation in healthy subjects by using a prime-boost regimen of DNA- and modied vaccinia virus Ankara-vectored vaccines expressing HIV-1 Gag coupled to CD8+ T-cell epitopes. J Virol 2006;80:471728. [13] Keefer MC, Frey SE, Elizaga M, Metch B, De Rosa SC, Barroso PF, et al. A phase I trial of preventive HIV vaccination with heterologous poxviral-vectors containing matching HIV-1 inserts in healthy HIV-uninfected subjects. Vaccine 2011;29:194858. [14] Ramanathan VD, Kumar M, Mahalingam J, Sathyamoorthy P, Narayanan PR, Solomon S, et al. A Phase 1 study to evaluate the safety and immunogenicity of a recombinant HIV type 1 subtype C-modied vaccinia Ankara virus vaccine candidate in Indian volunteers. AIDS Res Hum Retroviruses 2009;25:110716. [15] Vasan S, Schlesinger SJ, Chen Z, Hurley A, Lombardo A, Than S, et al. 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We would specially thank Victor Snchez, Maite Garca and Ana M. Garca for their technical assistance. We are also grateful to Marta Sala, Cristina Casadess and Saray Corral Carretero for their assistance during the follow-up of this trial. We acknowledge the Spanish HIV BioBank integrated in the Spanish AIDS Research Network (RIS) and collaborating centres for the clinical samples provided. We thank EuroVacc Foundation for providing pooled peptides to HIV antigens. The clinical lot of MVA-B was produced under a good manufacturing practice (GMP) funding by EuroVacc grant. Special thanks to all volunteers participating in this study for their perseverance and dedication, without whom the phase I clinical trial would not have been made possible. Contributors: Felipe Garca, Juan Carlos Lpez Bernaldo de Quirs, Montserrat Plana and Mariano Esteban conceived and designed the study. Felipe Garca, Inaki Prez and Vicens DazBrito undertook the statistical analyses. Felipe Garca, Juan Carlos Lpez Bernaldo de Quirs, Jos Alcam, Montserrat Plana and Mariano Esteban drafted the manuscript. Carmen E. Gmez, Beatriz Perdiguero, Jose L. Njera, Victoria Jimnez, Juan Garca-Arriaza, Alberto C. Guardo, Vicens Daz-Brito, Matilde Snchez Conde, Nuria Gonzlez, Amparo Alvarez, Jos Luis Jimnez, Judit Pich, Joan Albert Arnaiz, Mara J. Maleno, Agathe Len and Mara Angeles Munoz-Fernndez contributed to the study design and data management. Carmen E. Gmez, Beatriz Perdiguero, Jose L. Njera, Victoria Jimnez, Juan Garca-Arriaza, Alberto C. Guardo, Nuria Gonzlez, Amparo Alvarez, Jos Alcam, Montserrat Plana and Mariano Esteban performed the immune monitoring (cellular and humoral immunogenicity). Mara Angeles Munoz-Fernndez, Peter Liljestrm, Jonathan Weber, Giuseppe Pantaleo and Jos M Gatell participated in study analyses, manuscript preparation and revised it critically for important intellectual content. All authors reviewed and approved the nal version of the manuscript. Conict of interest statement: The authors do not have a commercial or other association that might pose a conict of interest. References
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