AF03 adjuvant improves anti-hemagglutinin and anti-neuraminidase immune responses induced by licensed seasonal quadrivalent influenza vaccines in mice.

Seasonal influenza remains a serious public health concern as the viral infection spreads easily from person to person and due to antigenic drift of neutralizing epitopes. Vaccination is the best method for disease prevention, however current seasonal influenza vaccines stimulate antibodies which are often effective against only antigenically similar strains. To boost the immune responses and increase vaccine effectiveness, adjuvants have been used for the past 20 years. The current study explores the use of oil-in-water adjuvant, AF03 to improve an immunogenicity of 2 licensed vaccines. A standard-dose inactivated quadrivalent influenza vaccine (IIV4-SD), containing both hemagglutinin (HA) and neuraminidase (NA) antigens, and recombinant quadrivalent influenza vaccine (RIV4), containing only HA-antigen were adjuvanted with AF03 in naïve BALB/c mouse model. Functional HA-specific antibody titers against all four homologous vaccine strains were enhanced by AF03, indicating potential increase in protective immunity. An increase in HA-specific total immunoglobulin G (IgG) binding titers were detected against homologous HAs, heterologous panel of 30 H3 HAs and seven Influenza B HAs. The neuraminidase inhibition (NAI) activity was significantly higher in IIV4-SD-AF03 group. Use of AF03 adjuvant improved the immune response to two influenza vaccines in a mouse model via an increase in functional and total antibodies against NA and a broad panel of HA-antigens.

Chimeric yellow fever 17D-Zika virus (ChimeriVax-Zika) as a live-attenuated Zika virus vaccine.

Zika virus (ZIKV) is an emerging mosquito-borne pathogen representing a global health concern. It has been linked to fetal microcephaly and other birth defects and neurological disorders in adults. Sanofi Pasteur has engaged in the development of an inactivated ZIKV vaccine, as well as a live chimeric vaccine candidate ChimeriVax-Zika (CYZ) that could become a preferred vaccine depending on future ZIKV epidemiology. This report focuses on the CYZ candidate that was constructed by replacing the pre-membrane and envelope (prM-E) genes in the genome of live attenuated yellow fever 17D vaccine virus (YF 17D) with those from ZIKV yielding a viable CYZ chimeric virus. The replication rate of CYZ in the Vero cell substrate was increased by using a hybrid YF 17D-ZIKV signal sequence for the prM protein. CYZ was highly attenuated both in mice and in human in vitro models (human neuroblastoma and neuronal progenitor cells), without the need for additional attenuating modifications. It exhibited significantly reduced viral loads in organs compared to a wild-type ZIKV and a complete lack of neuroinvasion following inoculation of immunodeficient A129 mice. A single dose of CYZ elicited high titers of ZIKV-specific neutralizing antibodies in both immunocompetent and A129 mice and protected animals from ZIKV challenge. The data indicate that CYZ is a promising vaccine candidate against ZIKV.

Single-dose vaccine against tick-borne encephalitis.

Tick-borne encephalitis (TBE) virus is the most important human pathogen transmitted by ticks in Eurasia. Inactivated vaccines are available but require multiple doses and frequent boosters to induce and maintain immunity. Thus far, the goal of developing a safe, live attenuated vaccine effective after a single dose has remained elusive. Here we used a replication-defective (single-cycle) flavivirus platform, RepliVax, to generate a safe, single-dose TBE vaccine. Several RepliVax-TBE candidates attenuated by a deletion in the capsid gene were constructed using different flavivirus backbones containing the envelope genes of TBE virus. RepliVax-TBE based on a West Nile virus backbone (RV-WN/TBE) grew more efficiently in helper cells than candidates based on Langat E5, TBE, and yellow fever 17D backbones, and was found to be highly immunogenic and efficacious in mice. Live chimeric yellow fever 17D/TBE, Dengue 2/TBE, and Langat E5/TBE candidates were also constructed but were found to be underattenuated. RV-WN/TBE was demonstrated to be highly immunogenic in Rhesus macaques after a single dose, inducing a significantly more durable humoral immune response compared with three doses of a licensed, adjuvanted human inactivated vaccine. Its immunogenicity was not significantly affected by preexisting immunity against WN. Immunized monkeys were protected from a stringent surrogate challenge. These results support the identification of a single-cycle TBE vaccine with a superior product profile to existing inactivated vaccines, which could lead to improved vaccine coverage and control of the disease.

Structural insights into the neutralization mechanism of a higher primate antibody against dengue virus.

The four serotypes of dengue virus (DENV-1 to -4) cause the most important emerging viral disease. Protein E, the principal viral envelope glycoprotein, mediates fusion of the viral and endosomal membranes during virus entry and is the target of neutralizing antibodies. However, the epitopes of strongly neutralizing human antibodies have not been described despite their importance to vaccine development. The chimpanzee Mab 5H2 potently neutralizes DENV-4 by binding to domain I of E. The crystal structure of Fab 5H2 bound to E from DENV-4 shows that antibody binding prevents formation of the fusogenic hairpin conformation of E, which together with in-vitro assays, demonstrates that 5H2 neutralizes by blocking membrane fusion in the endosome. Furthermore, we show that human sera from patients recovering from DENV-4 infection contain antibodies that bind to the 5H2 epitope region on domain I. This study, thus, provides new information and tools for effective vaccine design to prevent dengue disease.

Dengue virus neutralization is modulated by IgG antibody subclass and Fcgamma receptor subtype.

Severe dengue virus (DENV) infection is epidemiologically linked to pre-existing anti-DENV antibodies acquired by maternal transfer or primary infection. A possible explanation is that DENV immune complexes evade neutralization by engaging Fcgamma receptors (FcgammaR) on monocytes, natural targets for DENV in humans. Using epitope-matched humanized monoclonal antibodies (mAbs) and stable FcgammaR-transfected CV-1 cells, we found that DENV neutralization by IgG1, IgG3, and IgG4 mAbs was enhanced in high-affinity FcgammaRIA transfectants and diminished in low-affinity FcgammaRIIA transfectants, whereas neutralization by IgG2 mAbs (low-affinity ligands for both FcgammaRs) was diminished equally. In FcgammaR-negative Vero cells, IgG3 mAbs exhibited the strongest neutralizing activity and IgG2, the weakest. Our results demonstrate that DENV neutralization is modulated by the Fc region in an IgG subclass manner, likely through effects on virion and FcgammaR binding. Thus, the IgG antibody subclass profile generated by DENV infection or vaccination may independently influence the magnitude of the neutralizing response.

Humanized monoclonal antibodies derived from chimpanzee Fabs protect against Japanese encephalitis virus in vitro and in vivo.

Japanese encephalitis virus (JEV)-specific Fab antibodies were recovered by repertoire cloning from chimpanzees initially immunized with inactivated JE-VAX and then boosted with attenuated JEV SA14-14-2. From a panel of 11 Fabs recovered by different panning strategies, three highly potent neutralizing antibodies, termed Fabs A3, B2, and E3, which recognized spatially separated regions on the virion, were identified. These antibodies reacted with epitopes in different domains: the major determinant for Fab A3 was Lys(179) (domain I), that for Fab B2 was Ile(126) (domain II), and that for Fab E3 was Gly(302) (domain III) in the envelope protein, suggesting that these antibodies neutralize the virus by different mechanisms. Potent neutralizing antibodies reacted with a low number of binding sites available on the virion. These three Fabs and derived humanized monoclonal antibodies (MAbs) exhibited high neutralizing activities against a broad spectrum of JEV genotype strains. Demonstration of antibody-mediated protection of JEV infection in vivo is provided using the mouse encephalitis model. MAb B2 was most potent, with a 50% protective dose (ED(50)) of 0.84 microg, followed by MAb A3 (ED(50) of 5.8 microg) and then MAb E3 (ED(50) of 24.7 microg) for a 4-week-old mouse. Administration of 200 microg/mouse of MAb B2 1 day after otherwise lethal JEV infection protected 50% of mice and significantly prolonged the average survival time compared to that of mice in the unprotected group, suggesting a therapeutic potential for use of MAb B2 in humans.

Epitope determinants of a chimpanzee dengue virus type 4 (DENV-4)-neutralizing antibody and protection against DENV-4 challenge in mice and rhesus monkeys by passively transferred humanized antibody.

The chimpanzee monoclonal antibody (MAb) 5H2 is specific for dengue virus type 4 (DENV-4) and neutralizes the virus at a high titer in vitro. The epitope detected by the antibody was mapped by sequencing neutralization escape variants of the virus. One variant contained a Lys174-Glu substitution and another contained a Pro176-Leu substitution in domain I of the DENV-4 envelope protein (E). These mutations reduced binding affinity for the antibody 18- to >100-fold. Humanized immunoglobulin G (IgG) 5H2, originally produced from an expression vector, has been shown to be a variant containing a nine-amino-acid deletion in the Fc region which completely ablates antibody-dependent enhancement of DENV replication in vitro. The variant MAb, termed IgG 5H2 deltaD, is particularly attractive for exploring its protective capacity in vivo. Passive transfer of IgG 5H2 deltaD at 20 microg/mouse afforded 50% protection of suckling mice against challenge with 25 50% lethal doses of mouse neurovirulent DENV-4 strain H241. Passive transfer of antibody to monkeys was conducted to demonstrate proof of concept for protection against DENV challenge. Monkeys that received 2 mg/kg of body weight of IgG 5H2 deltaD were completely protected against 100 50% monkey infectious doses (MID50) of DENV-4, as indicated by the absence of viremia and seroconversion. A DENV-4 escape mutant that contained a Lys174-Glu substitution identical to that found in vitro was isolated from monkeys challenged with 10(6) MID50 of DENV-4. This substitution was also present in all naturally occurring isolates belonging to DENV-4 genotype III. These studies have important implications for possible antibody-mediated prevention of DENV infection.

Monoclonal antibody-mediated enhancement of dengue virus infection in vitro and in vivo and strategies for prevention.

Infection with dengue virus (DENV) or any other flavivirus induces cross-reactive, but weakly neutralizing or nonneutralizing, antibodies that recognize epitopes involving the fusion peptide in the envelope glycoprotein. Humanized mAb IgG 1A5, derived from a chimpanzee, shares properties of cross-reactive antibodies. mAb IgG 1A5 up-regulated DENV infection by a mechanism of antibody-dependent enhancement (ADE) in a variety of Fc receptor-bearing cells in vitro. A 10- to 1,000-fold increase of viral yield in K562 cells, dependent on the DENV serotype, was observed over a range of subneutralizing concentrations of IgG 1A5. A significant increase of DENV-4 viremia titers (up to 100-fold) was also demonstrated in juvenile rhesus monkeys immunized with passively transferred dilutions of IgG 1A5. These results, together with earlier findings of ADE of DENV-2 infection by a polyclonal serum, establish the primate model for analysis of ADE. Considering the abundance of these cross-reactive antibodies, our observations confirm that significant viral amplification could occur during DENV infections in humans with prior infection or with maternally transferred immunity, possibly leading to severe dengue. Strategies to eliminate ADE were explored by altering the antibody Fc structures responsible for binding to Fc receptors. IgG 1A5 variants, containing amino acid substitutions from the Fc region of IgG2 or IgG4 antibodies, reduced but did not eliminate DENV-4-enhancing activity in K562 cells. Importantly, a 9-aa deletion at the N terminus of the CH(2) domain in the Fc region abrogated the enhancing activity.

Chimpanzee Fab fragments and a derived humanized immunoglobulin G1 antibody that efficiently cross-neutralize dengue type 1 and type 2 viruses.

Passive immunization with monoclonal antibodies from humans or nonhuman primates represents an attractive alternative to vaccines for prevention of illness caused by dengue viruses (DENV) and other flaviviruses, including the West Nile virus. In a previous study, repertoire cloning to recover Fab fragments from bone marrow mRNA of chimpanzees infected with all four DENV serotypes (dengue virus serotype 1 [DENV-1] to DENV-4) was described. In that study, a humanized immunoglobulin G1 (IgG1) antibody that efficiently neutralized DENV-4 was recovered and characterized. In this study, the phage library constructed from the chimpanzees was used to recover Fab antibodies against the other three DENV serotypes. Serotype-specific neutralizing Fabs were not identified. Instead, we recovered DENV-neutralizing Fabs that specifically precipitated the envelope protein and were cross-reactive with all four DENV serotypes. Three of the Fabs competed with each other for binding to DENV-1 and DENV-2, although each of these Fabs contained a distinct complementarity determining region 3 (CDR3)-H sequence. Fabs that shared an identical or nearly identical CDR3-H sequences cross-neutralized DENV-1 and DENV-2 at a similar high 50% plaque reduction neutralization test (PRNT(50)) titer, ranging from 0.26 to 1.33 microg/ml, and neutralized DENV-3 and DENV-4 but at a titer 10- to 20-fold lower. One of these Fabs, 1A5, also neutralized the West Nile virus most efficiently among other flaviviruses tested. Fab 1A5 was converted to a full-length antibody in combination with human sequences for production in mammalian CHO cells. Humanized IgG1 1A5 proved to be as efficient as Fab 1A5 for cross-neutralization of DENV-1 and DENV-2 at a titer of 0.48 and 0.95 microg/ml, respectively. IgG1 1A5 also neutralized DENV-3, DENV-4, and the West Nile virus at a PRNT(50) titer of approximately 3.2 to 4.2 microg/ml. This humanized antibody represents an attractive candidate for further development of immunoprophylaxis against DENV and perhaps other flavivirus-associated diseases.

Epitope determinants of a chimpanzee Fab antibody that efficiently cross-neutralizes dengue type 1 and type 2 viruses map to inside and in close proximity to fusion loop of the dengue type 2 virus envelope glycoprotein.

The epitope determinants of chimpanzee Fab antibody 1A5, which have been shown to be broadly reactive to flaviviruses and efficient for cross-neutralization of dengue virus type 1 and type 2 (DENV-1 and DENV-2), were studied by analysis of DENV-2 antigenic variants. Sequence analysis showed that one antigenic variant contained a Gly-to-Val substitution at position 106 within the flavivirus-conserved fusion peptide loop of the envelope protein (E), and another variant contained a His-to-Gln substitution at position 317 in E. Substitution of Gly(106)Val in DENV-2 E reduced the binding affinity of Fab 1A5 by approximately 80-fold, whereas substitution of His(317)Gln had little or no effect on antibody binding compared to the parental virus. Treatment of DENV-2 with beta-mercaptoethanol abolished binding of Fab 1A5, indicating that disulfide bridges were required for the structural integrity of the Fab 1A5 epitope. Binding of Fab 1A5 to DENV-2 was competed by an oligopeptide containing the fusion peptide sequence as shown by competition enzyme-linked immunosorbent assay. Both DENV-2 antigenic variants were shown to be attenuated, or at least similar to the parental virus, when evaluated for growth in cultured cells or for neurovirulence in mice. Fab 1A5 inhibited low pH-induced membrane fusion of mosquito C6/36 cells infected with DENV-1 or DENV-2, as detected by reduced syncytium formation. Both substitutions in DENV-2 E lowered the pH threshold for membrane fusion, as measured in a fusion-from-within assay. In the three-dimensional structure of E, Gly(106) in domain II and His(317) in domain III of the opposite E monomer were spatially close. From the locations of these amino acids, Fab 1A5 appears to recognize a novel epitope that has not been mapped before with a flavivirus monoclonal antibody.

Identification of chimpanzee Fab fragments by repertoire cloning and production of a full-length humanized immunoglobulin G1 antibody that is highly efficient for neutralization of dengue type 4 virus.

A safe and effective dengue vaccine is still not available. Passive immunization with monoclonal antibodies from humans or nonhuman primates represents an attractive alternative for the prevention of dengue virus infection. Fab monoclonal antibodies to dengue type 4 virus (DENV-4) were recovered by repertoire cloning of bone marrow mRNAs from an immune chimpanzee and analyzed for antigen binding specificity, V(H) and V(L) sequences, and neutralizing activity against DENV-4 in vitro. Fabs 5A7, 3C1, 3E4, and 7G4 were isolated from a library constructed from a chimpanzee following intrahepatic transfection with infectious DENV-4 RNA. Fabs 5H2 and 5D9, which had nearly identical V(H) sequences but varied in their V(L) sequences, were recovered from a library constructed from the same chimpanzee after superinfection with a mixture of DENV-1, DENV-2, and DENV-3. In radioimmunoprecipitation, Fab 5A7 precipitated only DENV-4 prM, and Fabs 3E4, 7G4, 5D9, and 5H2 precipitated DENV-4 E but little or no prM. Fab 3E4 and Fab 7G4 competed with each other for binding to DENV-4 in an enzyme-linked immunosorbent assay, as did Fab 3C1 and Fab 5A7. Fab 5H2 recognized an epitope on DENV-4 that was separate from the epitope(s) recognized by other Fabs. Both Fab 5H2 and Fab 5D9 neutralized DENV-4 efficiently with a titer of 0.24 to 0.58 micro g/ml by plaque reduction neutralization test (PRNT), whereas DENV-4-neutralizing activity of other Fabs was low or not detected. Fab 5H2 was converted to full-length immunoglobulin G1 (IgG1) by combining it with human sequences. The humanized chimpanzee antibody IgG1 5H2 produced in CHO cells neutralized DENV-4 strains from different geographical origins at a similar 50% plaque reduction (PRNT(50)) titer of 0.03 to 0.05 micro g/ml. The DENV-4 binding affinities were 0.42 nM for Fab 5H2 and 0.24 nM for full-length IgG1 5H2. Monoclonal antibody IgG1 5H2 may prove valuable for passive immunoprophylaxis against dengue virus in humans.

Diversity and evolution of the envelope gene of dengue virus type 1.

The genetic diversity and phylogenetic relationships of a collection of strains of dengue virus type 1 (DV-1), isolated from different parts of the world, were investigated. Phylogenetic trees derived from the complete sequence of the E gene of 44 strains suggested the existence of five genetic types defined by a maximum nucleotide divergence within each group of 6%. The 22 strains from America were classified into a single genetic type that included strains associated either with classical dengue or hemorrhagic dengue episodes. Using a maximum likelihood procedure based on a single rate with dated tips model and substitution rates calculated at the third codon position, evolution of the five DV-1 genotypes was shown to conform to a molecular clock. The average rate of evolution was estimated to be approximately 16.2 x 10(-4) substitutions/third codon position site/year. Using this estimate, divergence among the DV-1 genotypes was calculated to have occurred approximately 100 years ago. Very low average value of the ratio of nonsynonymous-to-synonymous nucleotide substitutions, relative to the respective sites (0.046), indicated that the evolution of the E gene of the DV-1 is subject mostly to purifying selection.