Immunogenicity of poxvirus-based vaccines against Nipah virus.

Nipah virus (NiV), an emerging zoonotic pathogen in Southeast Asia, is transmitted from Pteropus species of fruit bats to a wide range of species, including humans, pigs, horses, dogs, and cats. NiV has killed millions of animals and caused highly fatal human outbreaks since no vaccine is commercially available. This study characterized the immunogenicity and safety of poxvirus-based Nipah vaccines that can be used in humans and species responsible for NiV transmission. Mice were vaccinated with modified vaccinia Ankara (MVA) and raccoon pox (RCN) viral vectors expressing the NiV fusion (F) and glycoprotein (G) proteins subcutaneously (SC) and intranasally (IN). Importantly, both vaccines did not induce significant weight loss or clinical signs of disease while generating high circulating neutralizing antibodies and lung-specific IgG and IgA responses. The MVA vaccine saw high phenotypic expression of effector and tissue resident memory CD8ɑ+ T cells in lungs and splenocytes along with the expression of central memory CD8ɑ+ T cells in lungs. The RCN vaccine generated effector memory (SC) and tissue resident (IN) CD8ɑ+ T cells in splenocytes and tissue resident (IN) CD8ɑ+ T cells in lung cells. These findings support MVA-FG and RCN-FG viral vectors as promising vaccine candidates to protect humans, domestic animals, and wildlife from fatal disease outcomes and to reduce the global threat of NiV.

Immunogenicity and protective activity of mRNA vaccine candidates against yellow fever virus in animal models.

Despite the success of the widely used attenuated yellow fever (YF) vaccine, its global supply remains a substantial barrier to implementing vaccination campaigns in endemic regions and combating emerging epidemics. In A129 mice and rhesus macaques, we evaluated the immunogenicity and protective activity of messenger RNA (mRNA) vaccine candidates encapsulated in lipid nanoparticles, expressing the pre-membrane and envelope proteins or the non-structural protein 1 of YF virus. Vaccine constructs induced humoral and cell-mediated immune responses in mice, resulting in protection against lethal YF virus infection after passive administration of serum or splenocytes from vaccinated mice. Vaccination of macaques induced sustained high humoral and cellular immune responses for at least 5 months after the second dose. Our data demonstrate that these mRNA vaccine candidates can be considered an attractive addition to the licensed YF vaccine supply based on the induction of functional antibodies correlating with protection and T-cell responses; they could alleviate the limited supply of current YF vaccines, mitigating future YF epidemics.

Optimization in the expression of ASFV proteins for the development of subunit vaccines using poxviruses as delivery vectors.

African swine fever virus (ASFV) causes a highly contagious hemorrhagic disease that affects domestic pig and Eurasian wild boar populations. To date, no safe and efficacious treatment or vaccine against ASF is available. Nevertheless, there are several reports of protection elicited by experimental vaccines based on live attenuated ASFV and some levels of protection and reduced viremia in other approaches such as DNA, adenovirus, baculovirus, and vaccinia-based vaccines. Current ASF subunit vaccine research focuses mainly on delivering protective antigens and antigen discovery within the ASFV genome. However, due to the complex nature of ASFV, expression vectors need to be optimized to improve their immunogenicity. Therefore, in the present study, we constructed several recombinant MVA vectors to evaluate the efficiency of different promoters and secretory signal sequences in the expression and immunogenicity of the p30 protein from ASFV. Overall, the natural poxvirus PrMVA13.5L promoter induced high levels of both p30 mRNA and specific anti-p30 antibodies in mice. In contrast, the synthetic PrS5E promoter and the S E/L promoter linked to a secretory signal showed lower mRNA levels and antibodies. These findings indicate that promoter selection may be as crucial as the antigen used to develop ASFV subunit vaccines using MVA as the delivery vector.

Impact of Molecular Modifications on the Immunogenicity and Efficacy of Recombinant Raccoon Poxvirus-Vectored Rabies Vaccine Candidates in Mice.

Rabies is an ancient disease that is responsible for approximately 59,000 human deaths annually. Bats (Order Chiroptera) are thought to be the original hosts of rabies virus (RABV) and currently account for most rabies cases in wildlife in the Americas. Vaccination is being used to manage rabies in other wildlife reservoirs like fox and raccoon, but no rabies vaccine is available for bats. We previously developed a recombinant raccoonpox virus (RCN) vaccine candidate expressing a mosaic glycoprotein (MoG) gene that protected mice and big brown bats when challenged with RABV. In this study, we developed two new recombinant RCN candidates expressing MoG (RCN-tPA-MoG and RCN-SS-TD-MoG) with the aim of improving RCN-MoG. We assessed and compared in vitro expression, in vivo immunogenicity, and protective efficacy in vaccinated mice challenged intracerebrally with RABV. All three candidates induced significant humoral immune responses, and inoculation with RCN-tPA-MoG or RCN-MoG significantly increased survival after RABV challenge. These results demonstrate the importance of considering molecular elements in the design of vaccines, and that vaccination with either RCN-tPA-MoG or RCN-MoG confers adequate protection from rabies infection, and either may be a sufficient vaccine candidate for bats in future work.

mRNA Vaccine Protects against Zika Virus.

Zika virus (ZIKV), a mosquito-borne flavivirus, has recently triggered global concern due to severe health complications. In 2015, a large ZIKV outbreak occurred in the Americas and established a link between ZIKV and microcephaly in newborn babies, spontaneous abortion, persistent viremia, and Guillain-Barré syndrome. While antivirals are being developed and prevention strategies focus on vector control, a safe and effective Zika vaccine remains unavailable. Messenger RNA (mRNA) vaccine technology has arisen as a flexible, simplified, and fast vaccine production platform. Here, we report on an mRNA vaccine candidate that encodes the pre-membrane and envelope (prM-E) glycoproteins of ZIKV strain Brazil SPH2015 and is encapsulated in lipid nanoparticles (LNPs). Our ZIKV prM-E mRNA-LNP vaccine candidate induced antibody responses that protected in AG129 mice deficient in interferon (IFN) alpha/beta/gamma (IFN-α/ß/γ) receptors. Notably, a single administration of ZIKV prM-E mRNA-LNP protected against a lethal dose of ZIKV, while a two-dose strategy induced strong protective immunity. E-specific double-positive IFN-γ and TNF-α T-cells were induced in BALB/c mice after immunizations with a two-dose strategy. With the success of mRNA vaccine technology in facing the coronavirus (COVID-19) pandemic, our data support the development of prM-E RNActive® as a promising mRNA vaccine against Zika to counter future epidemics.

Neonatal Development in Prenatally Zika Virus-Exposed Infant Macaques with Dengue Immunity.

Infants exposed to Zika virus (ZIKV) prenatally may develop birth defects, developmental deficits, or remain asymptomatic. It is unclear why some infants are more affected than others, although enhancement of maternal ZIKV infection via immunity to an antigenically similar virus, dengue virus (DENV), may play a role. We hypothesized that DENV immunity may worsen prenatal ZIKV infection and developmental deficits in offspring. We utilized a translational macaque model to examine how maternal DENV immunity influences ZIKV-exposed infant macaque neurodevelopment in the first month of life. We inoculated eight macaques with prior DENV infection with ZIKV, five macaques with ZIKV, and four macaques with saline. DENV/ZIKV-exposed infants had significantly worse visual orientation skills than ZIKV-exposed infants whose mothers were DENV-naive, with no differences in motor, sensory or state control development. ZIKV infection characteristics and pregnancy outcomes did not individually differ between dams with and without DENV immunity, but when multiple factors were combined in a multivariate model, maternal DENV immunity combined with ZIKV infection characteristics and pregnancy parameters predicted select developmental outcomes. We demonstrate that maternal DENV immunity exacerbates visual orientation and tracking deficits in ZIKV-exposed infant macaques, suggesting that human studies should evaluate how maternal DENV immunity impacts long-term neurodevelopment.

Clinical Presentation and Serologic Response during a Rabies Epizootic in Captive Common Vampire Bats (Desmodus rotundus).

We report mortality events in a group of 123 common vampire bats (Desmodus rotundus) captured in México and housed for a rabies vaccine efficacy study in Madison, Wisconsin. Bat mortalities occurred in México and Wisconsin, but rabies cases reported herein are only those that occurred after arrival in Madison (n = 15). Bats were confirmed positive for rabies virus (RABV) by the direct fluorescent antibody test. In accordance with previous reports, we observed long incubation periods (more than 100 days), variability in clinical signs prior to death, excretion of virus in saliva, and changes in rabies neutralizing antibody (rVNA) titers post-infection. We observed that the furious form of rabies (aggression, hyper-salivation, and hyper-excitability) manifested in three bats, which has not been reported in vampire bat studies since 1936. RABV was detected in saliva of 5/9 bats, 2-5 days prior to death, but was not detected in four of those bats that had been vaccinated shortly after exposure. Bats from different capture sites were involved in two separate outbreaks, and phylogenetic analysis revealed differences in the glycoprotein gene sequences of RABV isolated from each event, indicating that two different lineages were circulating separately during capture at each site.

Fibropapillomatosis in a Green Sea Turtle ( Chelonia mydas) from the Southeastern Pacific.

Fibropapillomatosis is a neoplastic disease that afflicts sea turtles. Although it is disseminated worldwide, cases of the disease have not been reported in the southeastern Pacific region. We describe a case of fibropapillomatosis in a green sea turtle ( Chelonia mydas) during its rehabilitation at the Machalilla National Park Rehabilitation Center, Ecuador. Viral presence was confirmed by PCR, targeting fragments of the chelonid alphaherpesvirus 5 (ChHV5) unique long (UL) genes, UL27, UL28, and UL30. The amplicons were sequenced and included in a global phylogenetic analysis of the virus with other reported sequences from GenBank. Results showed that the available viral sequences segregated into five phylogeographic groups: western Atlantic and eastern Caribbean, central Pacific, western Pacific, Atlantic, and eastern Pacific groups. The concatenated ChHV5 sequences from Ecuador clustered with the eastern Pacific sequences.

Molecular epidemiology and phylogenetic analysis of human papillomavirus infection in women with cervical lesions and cancer from the coastal region of Ecuador / Epidemiología molecular y análisis filogenético de la infección por el virus del papiloma humano en mujeres con lesiones cervicales y cáncer en la región litoral del Ecuador

The aim of the present study was to gather information regarding the molecular epidemiology of Human papillomavirus (HPV) and related risk factors in a group of women with low- and high-grade cervical lesions and cancer from the coastal region of Ecuador. In addition, we studied the evolution of HPV variants from the most prevalent types and provided a temporal framework for their emergence, which may help to trace the source of dissemination within the region. We analyzed 166 samples, including 57 CIN1, 95 CIN2/3 and 14 cancer cases. HPV detection and typing was done by PCR-sequencing (MY09/MY11). HPV variants and estimation of the time to most recent common ancestor (tMRCA) was assessed through phylogeny and coalescence analysis. HPV DNA was found in 54.4% of CIN1, 74.7% of CIN2/3 and 78.6% of cancer samples. HPV16 (38.9%) and HPV58 (19.5%) were the most prevalent types. Risk factors for the development of cervical lesions/cancer were the following: three or more pregnancies (OR = 4.3), HPV infection (OR = 3.7 for high-risk types; OR = 3.5 for HPV16), among others. With regard to HPV evolution, HPV16 isolates belonged to lineages A (69%) and D (31%) whereas HPV58 isolates belonged only to lineage A. The period of emergence of HPV16 was in association with human populations (tMRCA = 91 052 years for HPV16A and 27000 years for HPV16D), whereas HPV58A preceded Homo sapiens evolution (322 257 years). This study provides novel data on HPV epidemiology and evolution in Ecuador, which will be fundamental in the vaccine era.

El objetivo del presente estudio fue aportar información sobre la epidemiología molecular del virus del papiloma humano (human papillomavirus [HPV]) y los factores de riesgo asociados al desarrollo de lesiones cervicales y cáncer en mujeres de la costa del Ecuador. Además, se estudiaron la evolución de las variantes de los HPV más prevalentes y el marco temporal de su emergencia, para ayudar a rastrear la fuente de dispersión en la región. Se analizaron 166 muestras, incluyendo 57 y 95 casos de neoplasia intraepitelial cervical tipo 1 (CIN1) y tipo 2/3 (CIN2/3), respectivamente, y 14 de casos de cáncer. La detección/tipificación de HPV se realizó por PCR-secuenciación (MY09/MY11). La caracterización de variantes y la datación del ancestro común más reciente (tMRCA) se realizaron mediante filogenia y coalescencia. Se encontró ADN de HPV en el 54,4% de las muestras de CIN1, el 74,7% de las muestras de CIN2/3 y el 78,6% de las muestras de cáncer. Los tipos HPV16 (38,9%) y HPV58 (19,5%) fueron los más frecuentes. Los factores de riesgo para el desarrollo de lesiones cervicales/cáncer fueron 3 o más embarazos (OR = 4,3) e infección por HPV (O = 3,7 para HPV de alto riesgo, OR = 3,5 para HPV16), entre otros. En cuanto a la evolución viral, los aislados del HPV16 pertenecían a los linajes A (69%) y D (31%), mientras que los aislados del HPV58 pertenecían únicamente al linaje A. El período de emergencia del HPV16 estuvo asociado a poblaciones humanas (tMRCA = 91.052 años para HPV16Ay 27.000 para HPV16D), mientras que el del HPV58A precedió a la evolución de Homo sapiens (322.257 años). Este estudio proporciona datos novedosos sobre la epidemiología y la evolución del HPV en Ecuador, los cuales serán fundamentales en la era de la vacuna.

Molecular epidemiology and phylogenetic analysis of human papillomavirus infection in women with cervical lesions and cancer from the coastal region of Ecuador.

The aim of the present study was to gather information regarding the molecular epidemiology of Human papillomavirus (HPV) and related risk factors in a group of women with low- and high-grade cervical lesions and cancer from the coastal region of Ecuador. In addition, we studied the evolution of HPV variants from the most prevalent types and provided a temporal framework for their emergence, which may help to trace the source of dissemination within the region. We analyzed 166 samples, including 57 CIN1, 95 CIN2/3 and 14 cancer cases. HPV detection and typing was done by PCR-sequencing (MY09/MY11). HPV variants and estimation of the time to most recent common ancestor (tMRCA) was assessed through phylogeny and coalescence analysis. HPV DNA was found in 54.4% of CIN1, 74.7% of CIN2/3 and 78.6% of cancer samples. HPV16 (38.9%) and HPV58 (19.5%) were the most prevalent types. Risk factors for the development of cervical lesions/cancer were the following: three or more pregnancies (OR=4.3), HPV infection (OR=3.7 for high-risk types; OR=3.5 for HPV16), among others. With regard to HPV evolution, HPV16 isolates belonged to lineages A (69%) and D (31%) whereas HPV58 isolates belonged only to lineage A. The period of emergence of HPV16 was in association with human populations (tMRCA=91052 years for HPV16A and 27000 years for HPV16D), whereas HPV58A preceded Homo sapiens evolution (322257 years). This study provides novel data on HPV epidemiology and evolution in Ecuador, which will be fundamental in the vaccine era.