The development of recombinant subunit envelope-based vaccines to protect against dengue virus induced disease

Challenges associated with the interference observed between the dengue virus components within early tetravalent live-attenuated vaccines led many groups to explore the development of recombinant subunit based vaccines. Initial efforts in the field were hampered by low yields and/or improper folding, but the use of the Drosophila S2 cell expression system provided a mechanism to overcome these limitations. The truncated dengue envelope proteins (DEN-80E) for all four dengue virus types are expressed in the S2 system at high levels and have been shown to maintain native-like conformation. The DEN-80E proteins are potent immunogens when formulated with a variety of adjuvants, inducing high titer virus neutralizing antibody responses and demonstrating protection in both mouse and non-human primate models. Tetravalent vaccine formulations have shown no evidence of immune interference between the four DEN-80E antigens in preclinical models. Based on the promising preclinical data, the recombinant DEN-80E proteins have now advanced into clinical studies. An overview of the relevant preclinical data for these recombinant proteins is presented in this review.     

Preclinical development of a dengue tetravalent recombinant subunit vaccine: Immunogenicity and protective efficacy in nonhuman primates

We describe here the preclinical development of a dengue vaccine composed of recombinant subunit carboxy-truncated envelope (E) proteins (DEN-80E) for each of the four dengue serotypes. Immunogenicity and protective efficacy studies in Rhesus monkeys were conducted to evaluate monovalent and tetravalent DEN-80E vaccines formulated with ISCOMATRIX™ adjuvant. Three different doses and two dosing regimens (0, 1, 2 months and 0, 1, 2, and 6 months) were evaluated in these studies. We first evaluated monomeric (DEN4-80E) and dimeric (DEN4-80EZip) versions of DEN4-80E, the latter generated in an attempt to improve immunogenicity. The two antigens, evaluated at 6, 20 and 100 μg/dose formulated with ISCOMATRIX™ adjuvant, were equally immunogenic. A group immunized with 20 μg DEN4-80E and Alhydrogel™ induced much weaker responses. When challenged with wild-type dengue type 4 virus, all animals in the 6 and 20 μg groups and all but one in the DEN4-80EZip 100 μg group were protected from viremia. Two out of three monkeys in the Alhydrogel™ group had breakthrough viremia. A similar study was conducted to evaluate tetravalent formulations at low (3, 3, 3, 6 μg of DEN1-80E, DEN2-80E, DEN3-80E and DEN4-80E respectively), medium (10, 10, 10, 20 μg) and high (50, 50, 50, 100 μg) doses. All doses were comparably immunogenic and induced high titer, balanced neutralizing antibodies against all four DENV. Upon challenge with the four wild-type DENV, all animals in the low and medium dose groups were protected against viremia while two animals in the high-dose group exhibited breakthrough viremia. Our studies also indicated that a 0, 1, 2 and 6 month vaccination schedule is superior to the 0, 1, and 2 month schedule in terms of durability. Overall, the subunit vaccine was demonstrated to induce strong neutralization titers resulting in protection against viremia following challenge even 8-12 months after the last vaccine dose.     

Dengue vaccine: WHO position paper,July 2016 – recommendations

This article presents the World Health Organization’s (WHO) recommendations on the use of dengue vaccine excerpted from the WHO position paper on dengue vaccine published in the Weekly epidemiological Record in July 2016 (Dengue vaccine: WHO position paper, 2016) [1]. The current document is the first WHO position paper on dengue vaccination and focuses primarily on the available evidence concerning the only dengue vaccine to have been registered by National Regulatory Authorities. The position paper gives consideration to the epidemiological features of the disease and assesses the potential use of the vaccine for public health benefits.Footnotes to this paper provide a number of core references including references to grading tables that assess the quality of the scientific evidence, and to the evidence-to-recommendation table. In accordance with its mandate to provide guidance to Member States on health policy matters, WHO issues a series of regularly updated position papers on vaccines and combinations of vaccines against diseases that have an international public health impact. These papers are concerned primarily with the use of vaccines in large-scale immunization programmes; they summarize essential background information on diseases and vaccines, and conclude with WHO’s current position on the use of vaccines in the global context. This paper reflects the recommendations of the WHO’s Strategic Advisory Group of Experts (SAGE) on immunization. Recommendations on the use of this dengue vaccine were discussed by SAGE in April 2016; evidence presented at that SAGE meeting can be accessed at: http://www.who.int/immunization/sage/previous/en/index.html.     

Plant expressed EtMIC2 is an effective immunogen in conferring protection against chicken coccidiosis

Coccidiosis is an economically important disease affecting poultry industry and remains one of the major problems globally. Developing a cost effective sub-unit vaccine may help mitigate loss in the industry. Here, we report expressing one of the microneme proteins, EtMIC2 from Eimeria tenella in tobacco using Agrobacterium-mediated transient expression. The ability of plant expressed recombinant EtMIC2 in eliciting both humoral and cell-mediated immune responses were measured in the immunized birds. The protective efficacy in the vaccinated birds against a homologous challenge was also evaluated. Birds immunized with plant expressed EtMIC2 showed good sero-conversion, reduced oocyst output and increased weight gain when compared to control birds. Our data indicate that use of plant expressed recombinant EtMIC2 in birds was safe and had the potential in imparting partial protection in chickens against homologous challenge.     

Novel formulations enhance the thermal stability of live-attenuated flavivirus vaccines

Thermal stability is important for the manufacture, distribution and administration of vaccines, especially in tropical developing countries, where particularly adverse field conditions exist. Current live-attenuated flavivirus vaccines exhibit relatively poor liquid stability in clinical settings, and clinicians are instructed to discard the yellow fever vaccine 1 h after reconstitution. We have identified novel combinations of excipients that greatly enhance the thermal stability of live-attenuated DEN-2 PDK-53-based flavivirus vaccine candidates. Liquid formulations comprising a sugar, albumin and a pluronic polymer minimized the loss of flavivirus infectious titer to less than 0.5 log₁₀ pfu after storage for at least 8 h at 37 °C, 7 days at room temperature or at least 11 weeks at 4 °C. Additionally, these formulations prevented reduction of viral infectivity after two freeze-thaw cycles of virus. Formulated candidate vaccines were readily lyophilized and reconstituted with minimal loss of viral titers. In mice, the formulations were safe and did not hinder the ability of the vaccine virus to generate a potent, protective immune response. These formulations provided significantly greater liquid-phase stability than has been reported previously for other flavivirus vaccine formulations. The enhanced thermal stability provided by the formulations described here will facilitate the effective distribution of flavivirus vaccines worldwide.     

Evaluation of protection induced by a dengue virus serotype 2 envelope domain III protein scaffold/DNA vaccine in non-human primates

We describe the preclinical development of a dengue virus vaccine targeting the dengue virus serotype 2 (DENV2) envelope domain III (EDIII). This study provides proof-of-principle that a dengue EDIII protein scaffold/DNA vaccine can protect against dengue challenge. The dengue vaccine (EDIII-E2) is composed of both a protein particle and a DNA expression plasmid delivered simultaneously via intramuscular injection (protein) and gene gun (DNA) into rhesus macaques. The protein component can contain a maximum of 60 copies of EDIII presented on a multimeric scaffold of Geobacillus stearothermophilus E2 proteins. The DNA component is composed of the EDIII portion of the envelope gene cloned into an expression plasmid. The EDIII-E2 vaccine elicited robust antibody responses to DENV2, with neutralizing antibody responses detectable following the first boost and reaching titers of greater than 1:100,000 following the second and final boost. Vaccinated and naïve groups of macaques were challenged with DENV2. All vaccinated macaques were protected from detectable viremia by infectious assay, while naïve animals had detectable viremia for 2–7 days post-challenge. All naïve macaques had detectable viral RNA from day 2–10 post-challenge. In the EDIII-E2 group, three macaques were negative for viral RNA and three were found to have detectable viral RNA post challenge. Viremia onset was delayed and the duration was shortened relative to naïve controls. The presence of viral RNA post-challenge corresponded to a 10–30-fold boost in neutralization titers 28 days post challenge, whereas no boost was observed in the fully protected animals. Based on these results, we determine that pre-challenge 50% neutralization titers of >1:6000 correlated with sterilizing protection against DENV2 challenge in EDIII-E2 vaccinated macaques. Identification of the critical correlate of protection for the EDIII-E2 platform in the robust non-human primate model lays the groundwork for further development of a tetravalent EDIII-E2 dengue vaccine.     

Development of a recombinant tetravalent dengue virus vaccine: Immunogenicity and efficacy studies in mice and monkeys

Truncated recombinant dengue virus envelope protein subunits (80E) are efficiently expressed using the Drosophila Schneider-2 (S2) cell expression system. Binding of conformationally sensitive antibodies as well as X-ray crystal structural studies indicate that the recombinant 80E subunits are properly folded native-like proteins. Combining the 80E subunits from each of the four dengue serotypes with ISCOMATRIX^® adjuvant, an adjuvant selected from a set of adjuvants tested for maximal and long lasting immune responses, results in high titer virus neutralizing antibody responses. Immunization of mice with a mixture of all four 80E subunits and ISCOMATRIX^® adjuvant resulted in potent virus neutralizing antibody responses to each of the four serotypes. The responses to the components of the tetravalent mixture were equivalent to the responses to each of the subunits administered individually. In an effort to evaluate the potential protective efficacy of the Drosophila expressed 80E, the dengue serotype 2 (DEN2-80E) subunit was tested in both the mouse and monkey challenge models. In both models protection against viral challenge was achieved with low doses of antigen in the vaccine formulation. In non-human primates, low doses of the tetravalent formulation induced good virus neutralizing antibody titers to all four serotypes and protection against challenge with the two dengue virus serotypes tested. In contrast to previous reports, where subunit vaccine candidates have generally failed to induce potent, protective responses, native-like soluble 80E proteins expressed in the Drosophila S2 cells and administered with appropriate adjuvants are highly immunogenic and capable of eliciting protective responses in both mice and monkeys. These results support the development of a dengue virus tetravalent vaccine based on the four 80E subunits produced in the Drosophila S2 cell expression system.     

Escherichia coli expressed flagellin C (FliC) of Salmonella Typhi improved the protective efficacy of YopE against plague infection

In the current antibiotic resistance scenario, vaccines may provide best defense against lethal bacterial diseases. So far, there is no ideal vaccine available against plague. Despite providing complete protection in small animal models, F1/LcrV based vaccine failed to provide ideal protection in non human primates. Here, we cloned, expressed and purified YopE of Yersinia pestis and flagellin C (FliC) of Salmonella Typhi. However the best possible protection needs the significant induction of IFN-γ and TNF-α. To determine the protective potential of the recombinant YopE alone or in formulation with FliC, Balb/C mice were immunized subcutaneously. The formulations were prepared with alum, a human compatible adjuvant. In our studies, the combination of YopE?+?FliC induced significantly strong humoral and cellular immune responses. A combination of YopE?+?FliC provided 83% protection whereas YopE alone provided only 50% against 100LD₅₀ of Y. pestis in a mouse model.     

Immunogenicity of recombinant VP2 proteins of all nine serotypes of African horse sickness virus

African horse sickness (AHS) is an equine disease with a mortality of up to 90% for susceptible horses. The causative agent AHS virus (AHSV) is transmitted by species of Culicoides. AHSV serogroup within the genus Orbivirus of the Reoviridae family consists of nine serotypes that show no or very limited cross-neutralization. Of the seven structural proteins (VP1-VP7) of AHSV, VP2 is the serotype specific protein, and the major target for neutralizing antibodies. In this report, recombinant VP2 proteins of all nine serotypes were expressed individually by the baculovirus expression system and the immunogenicity of each was studied by immunization of guinea pigs with single VP2 as well as with cocktails of VP2 proteins. Homologous neutralizing antibodies measured by 50% plaque reduction assay showed varying degrees (from 37 to 1365) of titers for different VP2 proteins. A low cross-neutralizing antibody titer was found for genetically related AHSV serotypes. Immunization with VP2 cocktails containing equal amounts of each of the VP2 proteins also triggered neutralizing antibodies albeit to lower titers (4-117) to each of the serotypes in the cocktail. This study is a first step to develop a VP2 subunit vaccine for AHS and our results indicate that VP2 subunit vaccines are feasible individually or in a multi-serotype cocktail.     

Development of DENVax: a chimeric dengue-2 PDK-53-based tetravalent vaccine for protection against dengue fever

Dengue. virus infection is the leading arboviral cause of disease worldwide. A vaccine is being developed based on the attenuated DEN-2 virus, DEN-2 PDK-53. In this review, we summarize the characteristics of the parent DEN-2 PDK-53 strain as well as the chimeric viruses containing the prM and E genes of DEN-1, DEN-3 or DEN-4 virus in the genetic backbone of the DEN-2 PDK-53 virus (termed DENVax). Tetravalent DENVax formulations containing cloned, fully sequenced isolates of the DEN-2 PDK-53 virus and the three chimeras have been evaluated for safety and efficacy in preclinical animal models. Based on the safety, immunogenicity and efficacy in preclinical studies, Phase 1 clinical testing of DENVax has been initiated.     

Development of a dual vaccine for prevention of Brucella abortus infection and Escherichia coli O157:H7 intestinal colonization

Zoonoses that affect human and animal health have an important economic impact. In the study now presented, a bivalent vaccine has been developed that has the potential for preventing the transmission from cattle to humans of two bacterial pathogens: Brucella abortus and Shiga toxin-producing Escherichia coli (STEC). A 66 kDa chimeric antigen, composed by EspA, Intimin, Tir, and H7 flagellin (EITH₇) from STEC, was constructed and expressed in B. abortus Δpgm vaccine strain (BabΔpgm). Mice orally immunized with BabΔpgm(EITH₇) elicited an immune response with the induction of anti-EITH₇ antibodies (IgA) that clears an intestinal infection of E. coli O157:H7 three times faster (t = 4 days) than mice immunized with BabΔpgm carrier strain (t = 12 days). As expected, mice immunized with BabΔpgm(EITH₇) strain also elicited a protective immune response against B. abortus infection. A Brucella-based vaccine platform is described capable of eliciting a combined protective immune response against two bacterial pathogens with diverse lifestyles—the intracellular pathogen B. abortus and the intestinal extracellular pathogen STEC.     

流行性感冒灭活亚单位疫苗和裂解疫苗的安全性和免疫原性的比较研究

目的：比较流感亚单位疫苗与裂解疫苗的安全性和免疫原性。方法：流感亚单位疫苗和裂解疫苗按随机双盲法分别接种249名和250名6～12岁健康儿童。于接种当日和接种后3天内观察局部反应和全身反应。用血凝抑制试验检测接种儿童免疫前后的血凝抑制抗体(HI)滴度，计算抗体4倍增长阳转率，免疫后的保护水平抗体(≥1：40)的免疫成功率，以及抗体几何平均滴度((GMT)值和增长倍数。比较流感亚单位疫苗和裂解疫苗的临床观察结果。结果：两种疫苗接种后均未见局部反应，发热反应率和中高度发热反应率亚单位疫苗低于裂解疫苗，两组的差异有显著的统计学意义。未见其他全身反应。对疫苗3个毒株的血清学检测结果显示：亚单位疫苗的阳转率为74．5％～95．1％，保护水平抗体的免疫成功率为94．2％～99．6％，抗体GMT增长倍数为5．4～21．2。裂解疫苗的阳转率为79．8％～97．8％，保护水平抗体的免疫成功率为96．4％～100．0％，抗体GMT、增长倍数为6．4～21．0。两种疫苗的免疫学效果相似，所见差异无显著的统计学意义。结论：流感亚单位疫苗和裂解疫苗接种6～12岁儿童后反应轻微，安全性良好，亚单位疫苗发热反应率低于裂解疫苗。两种疫苗的免疫原性良好，具有同样显著的免疫效果，可以推广使用。