First Phase I human clinical trial of a killed whole-HIV-1 vaccine: demonstration of its safety and enhancement of anti-HIV antibody responses

Abstract

Background: Vaccination with inactivated (killed) whole-virus particles has been used to prevent a wide range of viral diseases. However, for an HIV vaccine this approach has been largely negated due to inherent safety concerns, despite the ability of killed whole-virus vaccines to generate a strong, predominantly antibody-mediated immune response in vivo. HIV-1 Clade B NL4-3 was genetically modified by deleting the nef and vpu genes and substituting the coding sequence for the Env signal peptide with that of honeybee melittin signal peptide to produce a less virulent and more replication efficient virus. This genetically modified virus (gmHIV-1_NL4-3) was inactivated and formulated as a killed whole-HIV vaccine, and then used for a Phase I human clinical trial (Trial Registration: Clinical Trials NCT01546818). The gmHIV-1_NL4-3 was propagated in the A3.01 human T cell line followed by virus purification and inactivation with aldrithiol-2 and γ-irradiation. Thirty-three HIV-1 positive volunteers receiving cART were recruited for this observer-blinded, placebo-controlled Phase I human clinical trial to assess the safety and immunogenicity.

Results: Genetically modified and killed whole-HIV-1 vaccine, SAV001, was well tolerated with no serious adverse events. HIV-1_NL4-3-specific PCR showed neither evidence of vaccine virus replication in the vaccine virus-infected human T lymphocytes in vitro nor in the participating volunteers receiving SAV001 vaccine. Furthermore, SAV001 with adjuvant significantly increased the pre-existing antibody response to HIV-1 proteins. Antibodies in the plasma of vaccinees were also found to recognize HIV-1 envelope protein on the surface of infected cells as well as showing an enhancement of broadly neutralizing antibodies inhibiting tier I and II of HIV-1 B, D, and A subtypes.

Conclusion: The killed whole-HIV vaccine, SAV001, is safe and triggers anti-HIV immune responses. It remains to be determined through an appropriate trial whether this immune response prevents HIV infection.

Keywords: AIDS; Clinical trial; HIV; Immune responses; Killed whole-HIV vaccine; Neutralizing antibodies; Safety.

Fig. 1

Construction of nef-, vpu-, and glycoprotein signal peptide replaced HIV-1_NL4-3 (gmHIV-1_NL3-4) and characterization of the SAV001 vaccine virus: The fragment between BsmBI and BglII (from 104 to 263 nucleotides) of HIV-1 NL4-3 nef gene was deleted and the stop codon (TAG) was inserted (a). The coding region of HIV-1 env signal peptide (30 amino acids) was replaced by the coding sequence of honeybee melittin signal peptide (21 amino acids) [24] (c). The vpu gene was deleted as the result of the Env signal peptide gene replacement due to its overlapping reading frame in the upstream of env gene. The pNL4-3 M/dNef with env signal peptide replaced plasmid was transfected into A3.01 human T-lymphocytes and recovered the genetically modified HIV-1 NL4-3 (gmHIV-1_NL4-3) virus (b). The genetic modification was confirmed by RT-PCR using honeybee melittin-specific primer with modified nef gene-specific primer (d). Electron micrograph of SAV001 vaccine showing intact virion (e). Western blot analyses of SAV001 using HIV-1 p24 antiserum (The NIH AIDS Reagent Program) and goat anti-HIV-1 gp120 polyclonal antibody (BIODESIGN), respectively (f). Aggregation of CD4⁺ AA2 [g, h (arrow)], and A3.01 [i, j (arrow)], as a result of gp120 on the SAV001 vaccine virus binding to the cell surface receptors. Induction of anti-gp120 antibody in SAV001 vaccine immunized rat sera using the gp140 trimer by ELISA (k, l)

Fig. 2

Flow diagram. The study was a randomized, observer-blinded, placebo-controlled study of killed-whole HIV-1 vaccine (SAV001-H) with or without adjuvant administered intramuscularly. Patients were screened for participation in this study within 28 days before enrollment. A total of 33 patients were planned to be enrolled in this study and randomized into Groups 1 and 2 of 17 and 16 patients, respectively. Patients were randomly assigned in a 9:3 ratio to receive either a single injection of the active investigational product or placebo, each of which was administered either without (Group 1) or with (Group 2) adjuvant

Fig. 3

Absence of residual infectious HIV-1 in SAV001 vaccine after inactivation: Detection of HIV-1 proviral DNA in infected cells by PCR and sequence analysis of viruses isolated from HIV-1 positive volunteer study subjects by 454 pyrosequencing. Lanes 1 Passage 3 of SAV001 vaccine virus infected cell DNA; Lanes 2 Passage 5 of SAV001 vaccine virus infected cell DNA: Lanes 3 Passage 10 of SAV001 vaccine virus infected cell DNA; Lanes 4 Passage 3 of HIV-1 NL4-3 wild type virus infected cell DNA; Lanes 5 Passage 3 gmHIV-1_NL4-3 mutant virus infected cell DNA (a). Multisequence alignments of reads were constructed using MUSCLE (PMID: 15034147), and all phylogenetic analysis was performed with MEGA5 (PMID: 21546353). The sequence reads were used to generate phylogenetic trees for further analysis through alignment with the reference viral sequences including NL4-3, 1 or 2 strains from HIV-1 subtype A, B, C, D, and F (b, c). All numbers represent subject ID in S (SAV001 group); SA (SAV001 + Adjuvant group), P (Placebo group), and PA (Placebo + Adjuvant group). PA1001014 and S3001024 in b were randomly selected to show the detail data

Fig. 4

Humoral immune responses of SAV001 vaccine. The humoral immune response was evaluated by assessment of antibody titres against p24, p17, gp120, and gp41 by ELISA. Microtiter plates were coated with specific HIV-1 recombinant antigens (p24, p17, gp120, and gp41 derived from HIV-1 IIIB (Immuno Diagnostics, USA) and serial dilutions of subject sera reacted against the specific antigens. The plates were read on the plate reader at 450 nm. Levels of antibodies in subjects from the entry and from week 4 to week 52 after vaccination were analyzed; p24 antibody (a), p17 antibody (b), gp120 antibody (c), and gp41 antibody (d). Numbers in the x-axis represent entry (E) and weeks after vaccination

Fig. 5

SAV001 elicits antibodies that recognize HIV-1 envelope glycoproteins at the surface of infected cells. CEM.NKr cells infected with full-length NL4.3 GFP ADA Env (WT) (a, c, e and g) or deleted for nef and vpu (N-U-) (b, d, f and h) were stained at 48 h post-infection with 1/1000 dilution of HIV + sera from Placebo- (a and b), Placebo + Adjuvant- (c and d), SAV001- (e and f) or SAV001 + Adjuvant- (g and h) vaccinees and then fluorescently labeled with an Alexa-Fluor-647 conjugated anti-human IgG secondary Ab [27, 28, 36]. Shown is the mean fluorescent intensity (MFI) of staining by sera from study participants. Statistical significance was tested using a paired t test (*p < 0.05)

Fig. 6

Neutralizing antibody response after vaccination. The analysis of the neutralizing antibodies before (green bar) and 4 weeks after (red bar) vaccination were carried out with 1:30 dilutions of sera and luciferase reporter gene expression was determined with Britelite (Perkin Elmer) using a Victor V plate reader (Perkin Elmer) (a). Neutralization activity of serial dilutions of high neutralizing antibodies of participants’ sera (red numbers on Fig. 5a) at both pre- and post-vaccination, using PRB926-04.A9.4237 (subtype B) and Q168ENVa2 (subtype A). Each data point represents the average IC50 of a single serum sample tested with the either virus, and the data from pre- and post-vaccination samples were paired for comparison (b). NA not available