Eveline L. Tierney

Temperature-sensitive mutants of influenza virus: VI. Transfer of ts lesions from the Asian subtype of influenza a virus (H2N2) to the Hong Kong subtype (H3N2)

Abstract Temperature-sensitive genetic lesions were transferred from the ts -1 (H2N2) and ts -2 (H2N2) mutants of influenza A virus to wild-type influenza A (H3N2) virus by genetic reassortment. The ts -2 (H3N2) recombinants appeared to be homogeneous and did not undergo recombination with one another, suggesting that the original ts -2 mutant of influenza A (H2N2) contained a ts lesion(s) on only one RNA segment of its genome. In contrast the ts -1 (H3N2) recombinants fell into three phenotypic subsets which differed in degree of temperature sensitivity. Initially, the three subsets of ts -1 (H3N2) recombinants were thought to represent distinct complementation-recombination groups. However, complementation-recombination between the three subsets of ts -1 (H3N2) recombinants subsets was variable. Subsequent study indicated that mutants in each of the three subsets shared one ts lesion and two of the subsets shared an additional ts lesion. The mechanism whereby viruses of the three subsets which share one or two ts lesions, and nevertheless undergo apparent complementation-recombination on occasion is not understood.

Cold-passaged human parainfluenza type 3 viruses contain ts and non-ts mutations leading to attenuation in rhesus monkeys.

Cold-passaged (CP) mutants derived from the JS strain of wild type wt parainfluenza type 3 virus (PIV3) are being evaluated as candidate live virus vaccines. The wt virus was serially passaged 45 times at low temperature and mutant clones with the cold-adapted (CA), temperature-sensitive (ts), and attenuation (ATT) phenotypes were selected following passage levels 12, 18 and 45 (cp12, cp18, and cp45). The cp45 virus was more ts than the cp12 or cp18 mutants, although all 3 mutant viruses were clearly attenuated in rhesus monkeys compared to wild type virus. The mean peak titers of the cp12 and cp18 viruses administered by the intratracheal route were at least 6000-fold lower than JSwt in both the upper and lower respiratory tracts. The cp45 virus was not recovered from monkeys administered virus by the i.t. route alone; however, when the cp45 virus was administered by the intranasal route, it replicated in the upper respiratory tract to a level comparable to that of the cp12 and cp18 viruses, but continued to be markedly restricted in the lower respiratory tract. These data indicate that the cp12 and cp18 viruses contain predominantly non-ts attenuating mutations whereas the cp45 mutant has both non-ts and ts attenuating mutations. Each of the CP mutants induced a high level of resistance to wild type virus challenge. Also, the ATT phenotype of the cp12 and cp18 viruses as measured in rhesus monkeys was stable after replication in chimpanzees or humans, respectively, although the ts phenotype was not. Based on its greater level of temperature sensitivity in vitro and its greater degree of attenuation in rhesus monkeys, the cp45 virus appears to be the most promising vaccine candidate for humans.

A Phase 1 Trial of MSP2-C1, a Blood-Stage Malaria Vaccine Containing 2 Isoforms of MSP2 Formulated with Montanide® ISA 720

Background In a previous Phase 1/2b malaria vaccine trial testing the 3D7 isoform of the malaria vaccine candidate Merozoite surface protein 2 (MSP2), parasite densities in children were reduced by 62%. However, breakthrough parasitemias were disproportionately of the alternate dimorphic form of MSP2, the FC27 genotype. We therefore undertook a dose-escalating, double-blinded, placebo-controlled Phase 1 trial in healthy, malaria-naïve adults of MSP2-C1, a vaccine containing recombinant forms of the two families of msp2 alleles, 3D7 and FC27 (EcMSP2-3D7 and EcMSP2-FC27), formulated in equal amounts with Montanide® ISA 720 as a water-in-oil emulsion. Methodology/Principal Findings The trial was designed to include three dose cohorts (10, 40, and 80 µg), each with twelve subjects receiving the vaccine and three control subjects receiving Montanide® ISA 720 adjuvant emulsion alone, in a schedule of three doses at 12-week intervals. Due to unexpected local reactogenicity and concern regarding vaccine stability, the trial was terminated after the second immunisation of the cohort receiving the 40 µg dose; no subjects received the 80 µg dose. Immunization induced significant IgG responses to both isoforms of MSP2 in the 10 µg and 40 µg dose cohorts, with antibody levels by ELISA higher in the 40 µg cohort. Vaccine-induced antibodies recognised native protein by Western blots of parasite protein extracts and by immunofluorescence microscopy. Although the induced anti-MSP2 antibodies did not directly inhibit parasite growth in vitro, IgG from the majority of individuals tested caused significant antibody-dependent cellular inhibition (ADCI) of parasite growth. Conclusions/Significance As the majority of subjects vaccinated with MSP2-C1 developed an antibody responses to both forms of MSP2, and that these antibodies mediated ADCI provide further support for MSP2 as a malaria vaccine candidate. However, in view of the reactogenicity of this formulation, further clinical development of MSP2-C1 will require formulation of MSP2 in an alternative adjuvant. Trial Registration Australian New Zealand Clinical Trials Registry 12607000552482

Phase 1 Study of Two Merozoite Surface Protein 1 (MSP142) Vaccines for Plasmodium falciparum Malaria

Objectives: To assess the safety and immunogenicity of two vaccines, MSP142-FVO/Alhydrogel and MSP142-3D7/Alhydrogel, targeting blood-stage Plasmodium falciparum parasites. Design: A Phase 1 open-label, dose-escalating study. Setting: Quintiles Phase 1 Services, Lenexa, Kansas between July 2004 and November 2005. Participants: Sixty healthy malaria-naïve volunteers 18–48 y of age. Interventions: The C-terminal 42-kDa region of merozoite surface protein 1 (MSP142) corresponding to the two allelic forms present in FVO and 3D7 P. falciparum lines were expressed in Escherichia coli, refolded, purified, and formulated on Alhydrogel (aluminum hydroxide). For each vaccine, volunteers in each of three dose cohorts (5, 20, and 80 μg) were vaccinated at 0, 28, and 180 d. Volunteers were followed for 1 y. Outcome Measures: The safety of MSP142-FVO/Alhydrogel and MSP142-3D7/Alhydrogel was assessed. The antibody response to each vaccine was measured by reactivity to homologous and heterologous MSP142, MSP119, and MSP133 recombinant proteins and recognition of FVO and 3D7 parasites. Results: Anti-MSP142 antibodies were detected by ELISA in 20/27 (74%) and 22/27 (81%) volunteers receiving three vaccinations of MSP142-FVO/Alhydrogel or MSP142-3D7/Alhydrogel, respectively. Regardless of the vaccine, the antibodies were cross-reactive to both MSP142-FVO and MSP142-3D7 proteins. The majority of the antibody response targeted the C-terminal 19-kDa domain of MSP142, although low-level antibodies to the N-terminal 33-kDa domain of MSP142 were also detected. Immunofluorescence microscopy of sera from the volunteers demonstrated reactivity with both FVO and 3D7 P. falciparum schizonts and free merozoites. Minimal in vitro growth inhibition of FVO or 3D7 parasites by purified IgG from the sera of the vaccinees was observed. Conclusions: The MSP142/Alhydrogel vaccines were safe and well tolerated but not sufficiently immunogenic to generate a biologic effect in vitro. Addition of immunostimulants to the Alhydrogel formulation to elicit higher vaccine-induced responses in humans may be required for an effective vaccine.

Safety, Immunogenicity, and Efficacy of Prime-Boost Immunization with Recombinant Poxvirus FP9 and Modified Vaccinia Virus Ankara Encoding the Full-Length Plasmodium falciparum Circumsporozoite Protein

ABSTRACT Heterologous prime-boost immunization with DNA and various recombinant poxviruses encoding malaria antigens is capable of inducing strong cell-mediated immune responses and partial protection in human sporozoite challenges. Here we report a series of trials assessing recombinant fowlpox virus and modified vaccinia virus Ankara encoding the Plasmodium falciparum circumsporozoite protein in various prime-boost combinations, doses, and application routes. For the first time, these vaccines were administered intramuscularly and at doses of up to 5 × 108 PFU. Vaccines containing this antigen proved safe and induced modest immune responses but showed no evidence of efficacy in a sporozoite challenge.

A Phase 1 trial of PfCP2.9 : An AMA1/MSP1 chimeric recombinant protein vaccine for Plasmodium falciparum malaria

Apical Membrane Antigen 1 (AMA1) and Merozoite Surface Protein 1 (MSP1) were produced as a recombinant fusion protein and formulated with the adjuvant Montanide ISA 720 with the aim of replicating the structure present in the parasite protein. A previous trial with this construct demonstrated the vaccine was safe and immunogenic but was associated with injection site reactogenicity. This Phase 1a dose-escalating, double blind, randomized, controlled trial of PfCP2.9/Montanide ISA 720 was conducted to evaluate alternative dose levels and vaccination schedules, with a pre-formulated vaccine that had undergone more in-depth and frequent quality control and stability analysis. The trial was conducted in seventy healthy Chinese malaria-naïve volunteers between January 2006 and January 2007. The objective was to assess the safety, reactogenicity and immunogenicity of 5, 20 and 50microg of PfCP2.9/ISA 720 under 2 different schedules. The most common adverse event was injection site tenderness (53%). The frequency and severity of adverse events was similar in both vaccination schedules. Antibody responses were induced and remained elevated throughout the study in volunteers receiving vaccine (p<0.001). Although high antibody titers as measured by ELISA to the PfCP2.9 immunogen were observed, biological function of these antibodies was not reflected by the in vitro inhibition of parasite growth, and there was limited recognition of fixed parasites in an immunofluorescence assay. At all three dose levels and both schedules, this formulation of PfCP2.9/ISA 720 is well tolerated, safe and immunogenic; however no functional activity against the parasite was observed.

Temperature-sensitive mutants of influenza: VIII. Genetic and biological characterization of ts mutants of influenza virus A (H3N2) and their assignment to complementation groups

Abstract Fifteen ts mutants of influenza A virus were characterized genetically and assigned to at least seven complementation groups. Six of the mutants were recombinant viruses while nine were clones derived directly from mutagenized H3N2 virus. The genetic analysis used a complementation-recombination assay carried out directly on the tissue culture monolayer. The nine clones of virus derived directly from mutagenized H3N2 virus were characterized in vivo in hamsters with respect to their ability to replicate in the lungs and nasal turbinates and with respect to the appearance of phenotypically wild-type virus in those organs of the hamster.

Phase 1 safety and immunogenicity trial of the Plasmodium falciparum blood-stage malaria vaccine AMA1-C1/ISA 720 in Australian adults

A Phase 1 trial was conducted in malaria-naïve adults to evaluate the recombinant protein vaccine apical membrane antigen 1-Combination 1 (AMA1-C1) formulated in Montanide ISA 720 (SEPPIC, France), a water-in-oil adjuvant. Vaccinations were halted early due to a formulation issue unrelated to stability or potency. Twenty-four subjects (12 in each group) were enrolled and received 5 or 20 microg protein at 0 and 3 months and four subjects were enrolled and received one vaccination of 80 microg protein. After first vaccination, nearly all subjects experienced mild to moderate local reactions and six experienced delayed local reactions occurring at Day 9 or later. After the second vaccination, three subjects experienced transient grade 3 (severe) local reactions; the remainder experienced grade 1 or 2 local reactions. All related systemic reactogenicity was grade 1 or 2, except one instance of grade 3 malaise. Anti-AMA1-C1 antibody responses were dose dependent and seen following each vaccination, with mean antibody levels 2-3 fold higher in the 20 microg group compared to the 5 microg group at most time points. In vitro growth-inhibitory activity was a function of the anti-AMA1 antibody titer. AMA1-C1 formulated in ISA 720 is immunogenic in malaria-naïve Australian adults. It is reasonably tolerated, though some transient, severe, and late local reactions are seen.

Temperature-sensitive mutants of influenza virus: VII. Transfer of the ts-1[E] lesions to a wild-type influenza A virus with the HON1 surface antigens

Abstract The nature of the genetic defect(s) present in the influenza A/Hong Kong/1968-ts-1[E] (H3N2) candidate vaccine virus, was analyzed by transferring them by genetic recombination to a 1943 HON1 wild-type virus. Three classes of ts viruses bearing the HO hemagglutinin were identified. One class consisted of two clones of virus, R1 and R8, which underwent complementation-recombination with each other but not with the Hong Kong/1968 (H3N2)-ts-1[E] parent virus. The second class consisted of clones of virus, such as R4 and R11, that failed to undergo complementation-recombination with R1, with R8, or with the ts-1[E] parent. These data indicated that the parent Hong Kong ts-1[E] virus possessed two ts lesions that segregated independently of each other and were presumably on different segments of the influenza A virus genome. The third class of ts mutants consisted of a spontaneously occurring ts mutant, clone R9; this clone underwent complementation-recombination with the ts-1[E] parent and other HO-ts recombinants and was shown to belong to a complementation group different than clones R1 and R8. The ts defects present in the Hong Kong-ts-1[E] donor virus segregated independently of the genes that coded for the epidemiologically important surface antigens, i.e., the hemagglutinin and the neuraminidase glycoproteins. The implications of these findings for the development of a live attenuated influenza virus vaccine are discussed.

Characterization of the attenuating M and NP gene segments of the avian influenza A/Mallard/78 virus during in vitro production of avian-human reassortant vaccine viruses and after replication in humans and primates

A unique requirement for live attenuated reassortant influenza vaccines is the need to generate new reassortant vaccine viruses with the appearance of each new antigenic variant. Thus, the attenuation phenotype conferred by the attenuated donor influenza virus must remain genetically stable during the generation of each new reassortant vaccine virus. In this study we used nucleotide sequence analysis to evaluate the genetic stability of the attenuating M and NP genes of the avian influenza A/Mallard/NY/6750/78 attenuated donor virus during the in vitro generation and subsequent in vivo replication of avian-human (AH) influenza A reassortant vaccine viruses in monkeys and humans. Nucleotide sequence changes in the M and NP genes occurred at a rate of approximately 0.61 substitutions/1000 nt/reassortant during in vitro generation of four AH reassortant viruses. Only two nucleotide sequence changes occurred in the M and NP gene segments of four isolates of H1N1 or H3N2 AH vaccine viruses following 6-8 days of replication in seronegative children, and neither change affected amino acids previously identified as playing a potential role in attenuation. In addition, there were no changes in the nucleotide sequence of the M and NP genes of single gene AH reassortant viruses following five serial passages in squirrel monkeys. Finally, there was no change in the level or duration of replication of the single gene reassortant viruses in the upper or lower respiratory tract of monkeys following serial passage.(ABSTRACT TRUNCATED AT 250 WORDS)