Molecular engineering and plant expression of an immunoglobulin heavy chain scaffold for delivery of a dengue vaccine candidate.

In order to enhance vaccine uptake by the immune cells in vivo, molecular engineering approach was employed to construct a polymeric immunoglobulin G scaffold (PIGS) that incorporates multiple copies of an antigen and targets the Fc gamma receptors on antigen-presenting cells. These self-adjuvanting immunogens were tested in the context of dengue infection, for which there is currently no globally licensed vaccine yet. Thus, the consensus domain III sequence (cEDIII) of dengue glycoprotein E was incorporated into PIGS and expressed in both tobacco plants and Chinese Ovary Hamster cells. Purified mouse and human cEDIII-PIGS were fractionated by HPLC into low and high molecular weight forms, corresponding to monomers, dimers and polymers. cEDIII-PIGS were shown to retain important Fc receptor functions associated with immunoglobulins, including binding to C1q component of the complement and the low affinity Fcγ receptor II, as well as to macrophage cells in vitro. These molecules were shown to be immunogenic in mice, with or without an adjuvant, inducing a high level IgG antibody response which showed a neutralizing potential against the dengue virus serotype 2. The cEDIII-PIGS also induced a significant cellular immune response, IFN-γ production and polyfunctional T cells in both the CD4+ and CD8+ compartments. This proof-of-principle study shows that the potent antibody Fc-mediated cellular functions can be harnessed to improve vaccine design, underscoring the potential of this technology to induce and modulate a broad-ranging immune response.

Generation of a More Immunogenic Measles Vaccine by Increasing Its Hemagglutinin Expression.

UNLABELLED: Imported measles virus (MV) outbreaks are maintained by poor vaccine responders and unvaccinated people. A convenient but more immunogenic vaccination strategy would enhance vaccine performance, contributing to measles eradication efforts. We report here the generation of alternative pediatric vaccines against MV with increased expression of the H protein in the background of the current MV vaccine strain. We generated two recombinants: MVvac2-H2, with increased full-length H expression resulting in a 3-fold increase in H incorporation into virions, and MVvac2-Hsol, vectoring a truncated, soluble form of the H protein that is secreted into the supernatants of infected cells. Replication fitness was conserved despite the duplication of the H cistron for both vectors. The modification to the envelope of MVvac2-H2 conferred upon this virus a measurable level of resistance to in vitro neutralization by MV polyclonal immune sera without altering its thermostability. Most interestingly, both recombinant MVs with enhanced H expression were significantly more immunogenic than their parental strain in outbred mice, while MVvac2-H2 additionally proved more immunogenic after a single, human-range dose in genetically modified MV-susceptible mice. IMPORTANCE: Measles incidence was reduced drastically following the introduction of attenuated vaccines, but progress toward the eradication of this virus has stalled, and MV still threatens unvaccinated populations. Due to the contributions of primary vaccine failures and too-young-to-be-vaccinated infants to this problem, more immunogenic measles vaccines are highly desirable. We generated two experimental MV vaccines based on a current vaccine's genome but with enriched production of the H protein, the main MV antigen in provoking immunity. One vaccine incorporated H at higher rates in the viral envelope, and the other secreted a soluble H protein from infected cells. The increased expression of H by these vectors improved neutralizing responses induced in two small-animal models of MV immunogenicity. The enhanced immunogenicity of these vectors, mainly from the MV that incorporates additional H, suggests their value as potential alternative pediatric MV vaccines.

Broadly neutralizing immune responses against hepatitis C virus induced by vectored measles viruses and a recombinant envelope protein booster.

Hepatitis C virus (HCV) infection remains a serious public health problem worldwide. Treatments are limited, and no preventive vaccine is available. Toward developing an HCV vaccine, we engineered two recombinant measles viruses (MVs) expressing structural proteins from the prototypic HCV subtype 1a strain H77. One virus directs the synthesis of the HCV capsid (C) protein and envelope glycoproteins (E1 and E2), which fold properly and form a heterodimer. The other virus expresses the E1 and E2 glycoproteins separately, with each one fused to the cytoplasmic tail of the MV fusion protein. Although these hybrid glycoproteins were transported to the plasma membrane, they were not incorporated into MV particles. Immunization of MV-susceptible, genetically modified mice with either vector induced neutralizing antibodies to MV and HCV. A boost with soluble E2 protein enhanced titers of neutralizing antibody against the homologous HCV envelope. In animals primed with MV expressing properly folded HCV C-E1-E2, boosting also induced cross-neutralizating antibodies against two heterologous HCV strains. These results show that recombinant MVs retain the ability to induce MV-specific humoral immunity while also eliciting HCV neutralizing antibodies, and that anti-HCV immunity can be boosted with a single dose of purified E2 protein. The use of MV vectors could have advantages for pediatric HCV vaccination.

A recombinant measles virus unable to antagonize STAT1 function cannot control inflammation and is attenuated in rhesus monkeys.

Measles remains a leading cause of death worldwide among children because it suppresses immune function. The measles virus (MV) P gene encodes three proteins (P, V, and C) that interfere with innate immunity, controlling STAT1, STAT2, mda5, and perhaps other key regulators of immune function. We identified here three residues in the shared domain of the P and V proteins-tyrosine 110, valine 112, and histidine 115-that function to retain STAT1 in the cytoplasm and inhibit interferon transcription. This information was used to generate a recombinant measles virus unable to antagonize STAT1 function (STAT1-blind MV) differing only in these three residues from a wild-type strain of well-defined virulence. This virus was used to assess the relevance of P and V interactions with STAT1 for virulence in primates. When a group of six rhesus monkeys (Macaca mulatta) was inoculated intranasally with STAT1-blind MV, viremia was short-lived, and the skin rash and other clinical signs observed with wild-type MV were absent. The STAT1-blind virus less efficiently controlled the inflammatory response, as measured by enhanced transcription of interleukin-6 and tumor necrosis factor alpha in peripheral blood mononuclear cells from infected hosts. Importantly, neutralizing antibody titers and MV-specific T-cell responses were equivalent in hosts infected with either virus. These findings indicate that efficient MV interactions with STAT1 are required to sustain virulence in a natural host by controlling the inflammatory response against the virus. They also suggest that selectively STAT1-blind MV may have utility as vectors for targeted oncolysis and vaccination.

Measles virus selectively blind to signaling lymphocytic activation molecule (SLAM; CD150) is attenuated and induces strong adaptive immune responses in rhesus monkeys.

The signaling lymphocytic activation molecule (SLAM; CD150) is the immune cell receptor for measles virus (MV). To assess the importance of the SLAM-MV interactions for virus spread and pathogenesis, we generated a wild-type IC-B MV selectively unable to recognize human SLAM (SLAM-blind). This virus differs from the fully virulent wild-type IC-B strain by a single arginine-to-alanine substitution at amino acid 533 of the attachment protein hemagglutinin and infects cells through SLAM about 40 times less efficiently than the isogenic wild-type strain. Ex vivo, this virus infects primary lymphocytes at low levels regardless of SLAM expression. When a group of six rhesus monkeys (Macaca mulatta) was inoculated intranasally with the SLAM-blind virus, no clinical symptoms were documented. Only one monkey had low-level viremia early after infection, whereas all the hosts in the control group had high viremia levels. Despite minimal, if any, viremia, all six hosts generated neutralizing antibody titers close to those of the control monkeys while MV-directed cellular immunity reached levels at least as high as in wild-type-infected monkeys. These findings prove formally that efficient SLAM recognition is necessary for MV virulence and pathogenesis. They also suggest that the selectively SLAM-blind wild-type MV can be developed into a vaccine vector.

Protective anti-hepatitis B virus responses in rhesus monkeys primed with a vectored measles virus and boosted with a single dose of hepatitis B surface antigen.

The widely used hepatitis B virus (HBV) vaccine is based on three doses of hepatitis B surface antigen (HBsAg) protein. We previously showed that vectored measles viruses (MV) expressing HBsAg retain measles vaccine function in monkeys but do not induce a protective anti-HBs response in all animals. We show here that a single dose of HBsAg protein following a three-dose vaccination regimen with an optimized HBsAg-expressing MV elicits protective anti-HBs responses in all four vaccinated Rhesus monkeys. Vaccination strategies coupling the effective, long-term immunity elicited by the high-coverage MV vaccine to prophylactic HBV immunity are discussed.

Heat shock effect upon dengue virus replication into U937 cells.

The molecules involved in dengue virus entry into human cells are currently unknown. We have previously shown that two surface heat shock proteins (Hsps), Hsp90 and Hsp70 are part of a receptor complex in monocytic cells. In the present report, the effect of heat shock (HS) on dengue virus infection is analyzed. We have documented a more than twofold increase in dengue virus infectivity after HS treatment in monocytic cells U937; this effect correlates mainly with an increase in viral entry due to a major presence of both Hsps on the surface of monocytic cells, particularly in membrane microdomains. Interestingly, since heat shock treatment at 6h post-infection also increased viral yields, it is likely that HS also modulates positively dengue virus replication.

Evidence that the 45-kD glycoprotein, part of a putative dengue virus receptor complex in the mosquito cell line C6/36, is a heat-shock related protein.

Dengue virus (DENV) is transmitted to humans by mosquitoes of the genus Aedes. Although several molecules have been described as part of DENV receptor complex in mosquito cells, none of them have been identified. Our group characterized two glycoproteins (40 and 45 kD) as part of the DENV receptor complex in C6/36 cells. Because identification of the mosquito cell receptor has been unsuccessful and some cell receptors described for DENV in mammalian cells are heat-shock proteins (HSPs), the role of HSPs in DENV binding and infection in C6/36 cells was evaluated. Our results indicate that gp45 and a 74-kD molecule (p74), which interact with DENV envelope protein, are immunologically related to HSP90. Although p74 is induced by heat shock, gp45 apparently is not. However, these proteins are relocated to the cell surface after heat-shock treatment, causing an increase in virus binding without any effect on virus yield.

A Recombinant Measles Vaccine with Enhanced Resistance to Passive Immunity.

Current measles vaccines suffer from poor effectiveness in young infants due primarily to the inhibitory effect of residual maternal immunity on vaccine responses. The development of a measles vaccine that resists such passive immunity would strongly contribute to the stalled effort toward measles eradication. In this concise communication, we show that a measles virus (MV) with enhanced hemagglutinin (H) expression and incorporation, termed MVvac2-H2, retained its enhanced immunogenicity, previously established in older mice, when administered to very young, genetically modified, MV-susceptible mice in the presence of passive anti-measles immunity. This immunity level mimics the sub-neutralizing immunity prevalent in infants too young to be vaccinated. Additionally, toward a more physiological small animal model of maternal anti-measles immunity interference, we document vertical transfer of passive anti-MV immunity in genetically-modified, MV susceptible mice and show in this physiological model a better MVvac2-H2 immunogenic profile than that of the parental vaccine strain. In sum, these data support the notion that enhancing MV hemagglutinin incorporation can circumvent in vivo neutralization. This strategy merits additional exploration as an alternative pediatric measles vaccine.

Novel vaccination approach for dengue infection based on recombinant immune complex universal platform.

Dengue infection is on the rise in many endemic areas of the tropics. Vaccination remains the most realistic strategy for prevention of this potentially fatal viral disease but there is currently no effective vaccine that could protect against all four known serotypes of the dengue virus. This study describes the generation and testing of a novel vaccination approach against dengue based on recombinant immune complexes (RIC). We modelled the dengue RIC on the existing Ebola RIC (Phoolcharoen, et al. Proc Natl Acad Sci USA 2011;108(Dec (51)):20695) but with a key modification that allowed formation of a universal RIC platform that can be easily adapted for use for other pathogens. This was achieved by retaining only the binding epitope of the 6D8 ant-Ebola mAb, which was then fused to the consensus dengue E3 domain (cEDIII), resulting in a hybrid dengue-Ebola RIC (DERIC). We expressed human and mouse versions of these molecules in tobacco plants using a geminivirus-based expression system. Following purification from the plant extracts by protein G affinity chromatography, DERIC bound to C1q component of complement, thus confirming functionality. Importantly, following immunization of mice, DERIC induced a potent, virus-neutralizing anti-cEDIII humoral immune response without exogenous adjuvants. We conclude that these self-adjuvanting immunogens have the potential to be developed as a novel vaccine candidate for dengue infection, and provide the basis for a universal RIC platform for use with other antigens.

Isolation of putative dengue virus receptor molecules by affinity chromatography using a recombinant E protein ligand.

Nucleotide sequences coding for the full-length envelope (E) glycoprotein gene of dengue virus type 4 was amplified using an RT-PCR method from infected C6/36 cells and cloned into pPROEx-Hta expression vector. The expression of the recombinant E protein in Escherichia coli was confirmed by Western blot using a polyclonal anti-dengue polyclonal antibody. The His-tagged fusion protein was obtained from the bacterial cellular extracts in almost pure form by immobilized metal affinity chromatography and the recombinant protein retained its ability to bind to 40 and 45 kDa proteins, previously described as putative receptors for dengue virus in C6/36 cells. To purify the 40 and 45 kDa molecules, a total protein extract from C6/36 cells was passed through an affinity chromatography column using immobilized recombinant E protein. After washing with isotonic buffer, elution was accomplished using a high salt buffer. The two proteins obtained, with molecular weights of 40 and 45 kDa, were recognized by dengue 4 virus, in virus overlay protein binding assay. This procedure allows further characterization of molecules that could be involved in dengue binding and entry.

Heat shock protein 90 and heat shock protein 70 are components of dengue virus receptor complex in human cells.

Dengue virus requires the presence of an unidentified cellular receptor on the surface of the host cell. By using a recently published affinity chromatography approach, an 84-kDa molecule, identified as heat shock protein 90 (HSP90) by matrix-assisted laser desorption ionization-time of flight mass spectrometry, was isolated from neuroblastoma and U937 cells. Based on the ability of HSP90 (84 kDa) to interact with HSP70 (74 kDa) on the surface of monocytes during lipopolysaccharide (LPS) signaling and evidence that LPS inhibits dengue virus infection, the presence of HSP70 was demonstrated in affinity chromatography eluates and by pull-down experiments. Infection inhibition assays support the conclusion that HSP90 and HSP70 participate in dengue virus entry as a receptor complex in human cell lines as well as in monocytes/macrophages. Additionally, our results indicate that both HSPs are associated with membrane microdomains (lipid rafts) in response to dengue virus infection. Moreover, methyl-beta-cyclodextrin, a raft-disrupting drug, inhibits dengue virus infection, supporting the idea that cholesterol-rich membrane fractions are important in dengue virus entry.

Cellular proteins from human monocytes bind to dengue 4 virus minus-strand 3' untranslated region RNA.

The synthesis of plus and minus RNA strands of several RNA viruses requires as a first step the interaction of some viral regulatory sequences with cellular and viral proteins. The dengue 4 virus genome, a single-stranded, positive-polarity RNA, is flanked by two untranslated regions (UTR) located in the 5' and 3' ends. The 3'UTR in the minus-strand RNA [3'UTR (-)] has been thought to function as a promoter for the synthesis of plus-strand RNA. To study the initial interaction between this 3'UTR and cellular and viral proteins, mobility shift assays were performed, and four ribonucleoprotein complexes (I through IV) were formed when uninfected and infected U937 cells (human monocyte cell line) interacted with the 3'UTR (-) of dengue 4 virus. Cross-linking assays with RNAs containing the complete 3'UTR (-) (nucleotides [nt] 101 to 1) or a partial sequence from nt 101 to 45 and nt 44 to 1 resulted in specific binding of some cellular proteins. Supermobility shift and immunoprecipitation assays demonstrated that the La protein forms part of these complexes. To determine the region in the 3' UTR that interacted with the La protein, two deletion mutants were generated. The mutant (del-96), with a deletion of nt 96 to 101, was unable to interact with the La protein, suggesting that La interacted with the 5' portion of the 3'UTR (-). Complex I, which was the main ribonucleoprotein complex formed with the 3'UTR (-) and which had the fastest electrophoretic migration, contained proteins such as calreticulin and protein disulfide isomerase, which constitute important components of the endoplasmic reticulum.