Robert Putnak

West Nile virus/dengue type 4 virus chimeras that are reduced in neurovirulence and peripheral virulence without loss of immunogenicity or protective efficacy

A candidate live attenuated vaccine strain was constructed for West Nile virus (WN), a neurotropic flavivirus that has recently emerged in the U.S. Considerable attenuation for mice was achieved by chimerization with dengue virus type 4 (DEN4). The genes for the structural premembrane and envelope proteins of DEN4 present in an infectious cDNA clone were replaced by the corresponding genes of WN strain NY99. Two of 18 cDNA clones of a WN/DEN4 chimera yielded full-length RNA transcripts that were infectious when transfected into susceptible cells. The two infectious clones shared a motif in the transmembrane signal domain located immediately downstream of the NS2B-NS3 protease cleavage site that separates the DEN4 capsid protein and the WN premembrane protein of the chimera. This motif, Asp and Thr at a position 3 and 6 amino acids downstream of the cleavage site, respectively, was not present in the 16 noninfectious cDNA clones. The WN/DEN4 chimera was highly attenuated in mice compared with its WN parent; the chimera was at least 28,500 times less neurovirulent in suckling mice inoculated intracerebrally and at least 10,000 times less virulent in adult mice inoculated intraperitoneally. Nonetheless, the WN/DEN4 chimera and a deletion mutant derived from it were immunogenic and provided complete protection against lethal WN challenge. These observations provide the basis for pursuing the development of a live attenuated WN vaccine.

Characterization of antibody responses to combinations of a dengue virus type 2 DNA vaccine and two dengue virus type 2 protein vaccines in rhesus macaques.

ABSTRACT We evaluated three nonreplicating dengue virus type 2 (DENV-2) vaccines: (i) a DNA vaccine containing the prM-E gene region (D), (ii) a recombinant subunit protein vaccine containing the B domain (i.e., domain III) of the E protein as a fusion with the Escherichia coli maltose-binding protein (R), and (iii) a purified inactivated virus vaccine (P). Groups of four rhesus macaques each were primed once and boosted twice using seven different vaccination regimens. After primary vaccination, enzyme-linked immunosorbent assay (ELISA) antibody levels increased most rapidly for groups inoculated with the P and DP combination, and by 1 month after the second boost, ELISA titers were similar for all groups. The highest plaque reduction neutralization test (PRNT) titers were seen in those groups that received the DR/DR/DR combination (geometric mean titer [GMT], 510), the P/P/P vaccine (GMT, 345), the DP/DP/DP combination (GMT, 287), and the R/R/R vaccine (GMT, 200). The next highest titers were seen in animals that received the D/R/R vaccine (GMT, 186) and the D/P/P vaccine (GMT, 163). Animals that received the D/D/D vaccine had the lowest neutralizing antibody titer (GMT, 49). Both ELISA and PRNT titers declined at variable rates. The only significant protection from viremia was observed in the P-vaccinated animals (mean of 0.5 days), which also showed the highest antibody concentration, including antibodies to NS1, and highest antibody avidity at the time of challenge.

A human challenge model for dengue infection reveals a possible protective role for sustained interferon gamma levels during the acute phase of illness.

Dengue has recently been defined by the World Health Organization as a major international public health concern. Although several vaccine candidates are in various stages of development, there is no licensed vaccine available to assist in controlling the further spread of this mosquito borne disease. The need for a reliable animal model for dengue disease increases the risk to vaccine developers as they move their vaccine candidates into large-scale phase III testing. In this paper we describe the cellular immune responses observed in a human challenge model for dengue infection; a model that has the potential to provide efficacy data for potential vaccine candidates in a controlled setting. Serum levels of sIL-2Rα and sTNF-RII were increased in volunteers who developed illness. Supernatants from in vitro stimulated PBMC were tested for cytokines associated with a T(H)1 or T(H)2 T-cell response (IL-2, TNF-α, IFN-γ, IL-4, IL-10, IL-5) and only IFN-γ was associated with protection against fever and/or viremia. Interestingly, IFN-γ levels drop to 0 pg/mL for volunteers who develop illness after challenge suggesting that some mechanism of immunosuppression may play a role in dengue illness. The human challenge model provides an opportunity to test potential vaccine candidates for efficacy prior to large-scale phase III testing, and hints at a possible mechanism for immune suppression by dengue.

Immunogenicity, safety, and tolerability of a recombinant measles-virus-based chikungunya vaccine: a randomised, double-blind, placebo-controlled, active-comparator, first-in-man trial

BACKGROUND Chikungunya is an emerging arthropod-borne disease that has spread from tropical endemic areas to more temperate climates of the USA and Europe. However, no specific treatment or preventive measure is yet available. We aimed to investigate the immunogenicity and safety of a live recombinant measles-virus-based chikungunya vaccine. METHODS We did a randomised, double-blind, placebo-controlled, active-comparator, phase 1, dose-escalation study at one centre in Vienna, Austria. Healthy men and women aged 18-45 years with no comorbidities were randomly assigned, by computer-generated block randomisation (block size of 14), to receive either one of three escalating doses of the measles-virus-based candidate vaccine (low dose [1·5 × 10(4) median tissue culture infection doses (TCID50) per 0·05 mL], medium dose [7·5 × 10(4) TCID50 per 0·25 mL], or high dose [3·0 × 10(5) TCID50 per 1·0 mL]), or the active comparator-Priorix. Participants were additionally block-randomised to receive a booster injection on either day 28 or day 90 after the first vaccination. Participants and study investigators were masked to group allocation. The primary endpoint was the presence of neutralising anti-chikungunya antibodies on day 28, as assessed by 50% plaque reduction neutralisation test. Analysis was by intention to treat and per protocol. This trial is registered with EudraCT, number 2013-001084-23. FINDINGS Between Nov 22, 2013, and Feb 25, 2014, we randomly assigned 42 participants to receive the low dose (n=12), the medium dose (n=12), or the high dose (n=12) of the measles-virus-based candidate vaccine, or Priorix (n=6), of whom 36 participants (86%; n=9, n=12, n=10, n=5, respectively) were included in the per-protocol population. The candidate vaccine raised neutralising antibodies in all dose cohorts after one immunisation, with seroconversion rates of 44% (n=4) in the low-dose group, 92% (n=11) in the medium-dose group, and 90% (n=10) in the high-dose group. The immunogenicity of the candidate vaccine was not affected by pre-existing anti-measles immunity. The second vaccination resulted in a 100% seroconversion for all participants in the candidate vaccine groups. The candidate vaccine had an overall good safety profile, and the rate of adverse events increased with vaccine dose and volume. No vaccination-related serious adverse events were recorded. INTERPRETATION The live recombinant measles-virus-based chikungunya vaccine had good immunogenicity, even in the presence of anti-vector immunity, was safe, and had a generally acceptable tolerability profile. This vaccine is the first promising measles-virus-based candidate vaccine for use in human beings. FUNDING Themis Bioscience GmBH.

Mapping of a region of dengue virus type-2 glycoprotein required for binding by a neutralizing monoclonal antibody

Abstract Envelope glycoprotein E of flaviviruses is exposed at the surface of the virion, and is responsible for eliciting a neutralizing antibody (Ab) response, as well as protective immunity in the host. In this report, we describe a method for the fine mapping of a linear sequence of the E protein of dengue virus type-2 (DEN-2), recognized by a type-specific and neutralizing monoclonal Ab (mAb), 3H5. First, an Escherichia coli expression vector containing a heat-inducible λ p l promoter was used to synthesize several truncated, and near-full length E polypeptides. Reactivities of these polypeptides with polyclonal mouse hyperimmune sera, as well as the 3H5 mAb revealed the location of the 3H5-binding site to be within a region of 166 amino acids (aa) between aa 255 and 422. For fine mapping, a series of targeted deletions were made inframe within this region using the polymerase chain reaction (PCR). The hydrophilicity pattern of this region was used as a guide to systematically delete the regions encoding the various groups of surface aa residues within the context of a near-full-length E polypeptide by using PCR. The 3H5-binding site was thus precisely mapped to a region encoding 12 aa (between aa 386 and 397). A synthetic peptide containing this sequence was able to bind to the 3H5 mAb specifically, as shown by enzyme-linked immunosorbent assay. In addition, we show that rabbit Abs raised against the synthetic peptide of 12 aa were able to bind to the authentic E protein, and to neutralize DEN-2 virus in a plaque reduction assay.

A Phase II, Randomized, Safety and Immunogenicity Study of a Re-Derived, Live-Attenuated Dengue Virus Vaccine in Healthy Adults

Two formulations of a new live tetravalent dengue virus (DENV) vaccine produced using re-derived master seeds from a precursor vaccine and that same precursor vaccine as a control were compared in a placebo-controlled, randomized, observer-blind, phase II trial of 86 healthy adults. Two vaccine doses were administered 6 months apart; a third dose was offered to a subset. Symptoms and signs of dengue-like illness reported after vaccination were mild to moderate, transient, and occurred with similar frequency among recipients of the new DENV vaccine and placebo, except for rash. Neither dengue nor vaccine-related serious adverse events were reported. The first DENV vaccine dose was moderately immunogenic; the second dose increased the potency and breadth of the neutralizing antibody response. Tetravalent response rates to the new formulations were 60% and 66.7% in unprimed subjects. A third dose did not increase tetravalent antibody rates. The new DENV vaccine candidates merit additional evaluation.

Protection against dengue virus by non-replicating and live attenuated vaccines used together in a prime boost vaccination strategy

A new vaccination strategy for dengue virus (DENV) was evaluated in rhesus macaques by priming with tetravalent purified inactivated virus (TPIV) or tetravalent plasmid DNA vaccines expressing the structural prME gene region (TDNA) then boosting 2 months later with a tetravalent live attenuated virus (TLAV) vaccine. Both vaccine combinations elicited virus neutralizing (N) antibodies. The TPIV/TLAV combination afforded complete protection against DENV 3 challenge at month 8. In a second experiment, priming with TPIV elicited N antibodies against all four serotypes (GMT 1:28 to 1:43). Boosting with TLAV led to an increase in the GMT for each serotype (1:500 to 1:1200 for DENVs 1, 3, and 4, and greater than 1:6000 for DENV 2), which declined by month 8 (GMT 1:62 for DENV 3, 1:154 for DENV 1, 1:174 for DENV 4, and 1:767 for DENV 2). After challenge with each one of the four DENV serotypes, vaccinated animals exhibited no viremia but showed anamnestic antibody responses to the challenge viruses.

Correlation of protection against Japanese encephalitis virus and JE vaccine (IXIARO®) induced neutralizing antibody titers☆

Immune sera from volunteers vaccinated in a blinded Phase 3 clinical trial with JE-VAX(®) and a new Japanese encephalitis virus (JEV) vaccine (IC51 or IXIARO), were tested for the ability to protect mice against lethal JEV challenge. Sera from IXIARO vaccinated subjects were pooled into four batches based on neutralizing antibody measured by plaque reduction neutralization test (PRNT(50) titer): high (∼200), medium (∼40-50), low (∼20) and negative (<10). Pooled sera from JE-VAX(®) vaccinated subjects (PRNT(50) titer∼55) and pooled JEV antibody negative pre-vaccination sera were used as controls. Groups of ten 6- to 7-week-old female ICR mice were injected intraperitoneally with 0.5 ml of each serum pool diluted 1:2 or 1:10, challenged approximately 18 h later with a lethal dose of either JEV strain SA14 (genotype III) or strain KE-093 (genotype I) and observed for 21 days. All mice in the non-immune serum groups developed clinical signs consistent with JEV infection or died, whereas high titer sera from both IXIARO and JE-VAX(®) sera protected 90-100% of the animals. Statistical tests showed similar protection against both JEV strains SA14 and KE-093 and protection correlated with the anti-JEV antibody titer of IXIARO sera as measured by PRNT(50). Ex vivo neutralizing antibody titers showed that almost all mice with a titer of 10 or greater were fully protected. In a separate study, analysis of geometric mean titers (GMTs) of the groups of mice vaccinated with different doses of IXIARO and challenged with JEV SA14 provided additional evidence that titers≥10 were protective.

Formalin-inactivated whole virus and recombinant subunit flavivirus vaccines.

The Flaviviridae is a family of arthropod-borne, enveloped, RNA viruses that contain important human pathogens such as yellow fever (YF), Japanese encephalitis (JE), tick-borne encephalitis (TBE), West Nile (WN), and the dengue (DEN) viruses. Vaccination is the most effective means of disease prevention for these viral infections. A live-attenuated vaccine for YF, and inactivated vaccines for JE and TBE have significantly reduced the incidence of disease for these viruses, while licensed vaccines for DEN and WN are still lacking despite a significant disease burden associated with these infections. This review focuses on inactivated and recombinant subunit vaccines (non-replicating protein vaccines) in various stages of laboratory development and human testing. A purified, inactivated vaccine (PIV) candidate for DEN will soon be evaluated in a phase 1 clinical trial, and a second-generation JE PIV produced using similar technology has advanced to phase 2/3 trials. The inactivated TBE vaccine used successfully in Europe for almost 30 years continues to be improved by additional purification, new stabilizers, an adjuvant, and better immunization schedules. The recent development of an inactivated WN vaccine for domestic animals demonstrates the possibility of producing a similar vaccine for human use. Advances in flavivirus gene expression technology have led to the production of several recombinant subunit antigen vaccine candidates in a variety of expression systems. Some of these vaccines have shown sufficient promise in animal models to be considered as candidates for evaluation in clinical trials. Feasibility of non-replicating flavivirus vaccines has been clearly demonstrated and further development is now warranted.

Interference and Facilitation Between Dengue Serotypes in a Tetravalent Live Dengue Virus Vaccine Candidate

BACKGROUND Live, multivalent vaccines have historically exhibited interference in humans; live dengue virus (DENV) vaccines have proven no exception. METHODS To characterize interactions between DENV serotypes in a tetravalent live-attenuated virus vaccine candidate, we analyzed data from a factorial clinical trial in which all combinations of high- and low-dose DENV serotypes were combined in 16 live-attenuated tetravalent vaccine formulations (N = 64) and administered to flavivirus-naive adult volunteers. Regression models considered the outcomes of reactogenicity and seroconversion, controlling for all serotype doses simultaneously. Additionally, results were compared against earlier evaluations of the same viruses administered as monovalent formulations. RESULTS DENV-1 was immunologically dominant in both monovalent and tetravalent formulations. In tetravalent formulations, DENV-1 and DENV-2 antagonized each other, with a high dose of one decreasing seroconversion to the other. However, high-dose DENV-1 significantly increased seroconversion against 3 or more serotypes, increasing seroconversion to DENV-1, DENV-3, and DENV-4. The highest reactogenicity occurred when DENV-1 was at high dose and all others were low; reactogenicity decreased with the incorporation of other high-dose serotypes. CONCLUSIONS Interference and facilitation occurred between serotypes in the live vaccine candidate evaluated. These analyses suggest that it may be possible to exploit facilitation to increase overall seroconversion.