Molecular Evidence of Human Monkeypox Virus Infection, Sierra Leone.

Monkeypox virus is a zoonotic disease endemic to Africa. In 2017, we confirmed a case of human monkeypox virus in Sierra Leone by molecular and serologic methods. Sequencing analysis indicated the virus belongs to the West African clade and data suggest it was likely transmitted by wild animals.

Monkeypox virus: past and present.

BACKGROUND: The objective of this paper is to analyze the current status of monkeypox worldwide. In the face of this public health threat, our purpose is to elucidate the clinical characteristics and epidemiology of monkeypox, the developmental progress of monkeypox-related drugs and the vaccines available. DATA SOURCES: The literature review was performed in databases including PubMed, Science Direct and Google Scholar up to July 2022. RESULTS: Since May 2022, the World Health Organization has reported more than 45,000 confirmed cases from 92 nonendemic countries, including nine deaths. Although some women and children have been infected so far, most cases have occurred among men who have sex with other men, especially those with multiple sexual partners or anonymous sex. CONCLUSIONS: Pediatric monkeypox infection has been associated with a higher likelihood of severe illness and mortality than in adults. Severe monkeypox illness in pediatrics often requires adjunctive antiviral therapy. It is crucial for all countries to establish sound monitoring and testing systems and be prepared with emergency preparedness.

[High expression of HPV16L2N120E7E6 fusion protein in E. coli and its inhibitory effect on tumor growth in mice].

OBJECTIVE: To investigate the high expression of HPV16L2N120E7E6 fusion protein by prokaryotic expression system, and evaluate its immunogenicity and antitumor efficacy in vaccinated mice. METHODS: The HPV16L2N120E7E6 fusion gene, its codons were optimized to increase the expression of the protein, was constructed by overlap extension PCR and inserted into prokaryotic expression vector pET9a. Then the fusion protein was expressed by inducing with IPTG in E. coli strain BL21 (DE3) harboring with plasmid pETL2N120E7E6, and further detected by SDS-PAGE and Western-blot. Finally, the humoral and cellular immune responses were measured by ELISA and ELISPOT, respectively, in vaccinated mice with the purified HPV16L2N120E7E6 fusion protein, and the antitumor efficacy was assessed in mice using the TC-1 tumor challenge model. RESULTS: The codon-optimized HPV16L2N120E7E6 fusion gene was highly expressed in E. coli strain BL21 (DE3) harboring with plasmid pETL2N120E7E6, and the amount of fusion protein was nearly 48.6% of the total bacterial protein. The purified fusion protein could induce high titer of specific antibody against L2, E7 and E6 in vaccinated mice. When accompanied with the adjuvant CpG, the fusion protein was able to elicit strong and moderate cellular immune responses in vaccinated mice against peptide HPV16E7(49-57) and peptide pools of HPV16E6, respectively. Furthermore, the tumor therapeutic experiment showed that HPV16L2N120E7E6 + CpG could prevent the tumor formation in 80.0% (8/10) vaccinated mice. CONCLUSIONS: The data of this study suggest that HPV16L2N120E7E6 fusion protein could be a promising candidate vaccine for treatment of chronic HPV16 infection and post-operative adjuvant therapy for cervical cancer.

Isolation and Characterization of Monkeypox Virus from the First Case of Monkeypox - Chongqing Municipality, China, 2022.

Introduction: The first imported case of monkeypox (MPX) from the mainland of China was reported in September 2022. Herein, the study reports the isolation and characterization of MPX virus (MPXV) in this case. Methods: Clinical specimens including skin blister fluid, oropharyngeal and nasopharyngeal swabs, and blood were collected and inoculated onto Vero cells. The isolated virus was identified as MPXV using quantitative polymerase chain reaction (qPCR), cytopathic effects (CPEs), immunofluorescence assay (IFA) and transmission electron microscopy (TEM). Plaque assays were employed to quantify infectious plaque-forming units (PFUs). The plaque reduction neutralization test (PRNT) was developed to determine the neutralizing antibody (nAb) against MPXV. Results: MPXV replication was confirmed with qPCR. Typical CPEs were observed 48 h post-incubation. The isolated virus was named MPXV-B.1-China-C-Tan-CQ01. IFA showed that MPXV reacted with serum of MPX case. Orthopoxvirus morphology was observed using TEM. The virus titer increased to >106 PFU/mL after three passages. The serum PRNT 50% neutralization titer (NT50) was 35 for the MPX patient 6 days after symptom onset. Discussion: The study successfully isolated the first MPXV strain in the mainland of China, MPXV-B.1-China-C-Tan-CQ01. Infectious titration and PRNT methods have been developed. The study provides key resources and technical platforms for further research as well as anti-viral drug and vaccine development against MPX.

Immunogenicity in mice and rhesus monkeys vaccinated with recombinant vaccinia virus expressing bivalent E7E6 fusion proteins from human papillomavirus types 16 and 18.

BACKGROUND: Persistent infection with high-risk human papillomavirus (HPV) is a predominant cause of cervical cancer, and HPV16 and HPV18 occur in 50% and 20% of cervical cancer cases, respectively. The viral oncogenes E6 and E7 are constitutively expressed by HPV-associated tumour cells and can therefore be used as target antigens for immunotherapy. In this study, we constructed a recombinant vaccinia virus co-expressing the HPV16/18 E7E6 fusion proteins (rVVJ16/18E7E6) for use as a therapeutic vaccine for the treatment of HPV16⁺ and HPV18⁺ cancers. METHODS: We constructed a bivalent recombinant vaccinia virus expressing modified E7E6 fusion proteins of HPV type 16 and 18 (rVVJ16/18E7E6) based on the vaccinia virus Tiantan strain. We then defined the cellular immune responses to the virus in mice and rhesus monkeys and assessed antitumour efficacy of these responses in mice using the TC-1 tumour challenge model. RESULTS: Our data demonstrated that rVVJ16/18E7E6 was able to elicit varying levels of CD8⁺ T cell immune responses and lysis of target cells in mice in response to peptides HPV16E749₋57 and HPV18E667₋75. Furthermore, the virus was also able to induce anti-tumour responses in the HPV16⁺ TC-1 tumour challenge model, including partial protection (30-40%) and delayed tumour appearance. In addition, the virus was able to induce immune responses in rhesus monkeys. CONCLUSIONS: The recombinant vaccinia virus rVVJ16/18E7E6 can generate clear and significant cellular immunity in both mice and rhesus monkeys. These data provide a basis for the use of this recombinant virus as a potential vaccine candidate for further study.

Non-replicating Vaccinia Virus TianTan Strain (NTV) Translation Arrest of Viral Late Protein Synthesis Associated With Anti-viral Host Factor SAMD9.

NTV is a highly attenuated virus that was created by genetically deleting 26 genes related to host range and virulence from TianTan strain. Since NTV is highly attenuated, it has been used widely as an optimizing viral vector. In this study, we explored the biological characteristics in vitro and the host restriction mechanism of NTV. Most cell lines do not support sufficient dissemination and replication of NTV, and in non-permissive cell line HeLa, the replication block of NTV occurred at the translation stage of viral late protein expression. Lack of PKR activity was not sufficient to rescue expression of viral late proteins and replication, even though the phosphorylation level of eIF2α increased in NTV-infected HeLa cells. Moreover, the translation inhibition of NTV in HeLa cells was dependent upon a SAMD9 signaling pathway, as demonstrated by silencing SAMD9 expression with siRNA and observing the colocalization of SAMD9 and AVGs. Reinserting C7L or K1L into NTV rescued the late viral protein expression and replication of NTV in HeLa cells. Among the genes deleted in NTV, C7L or/and K1L gene was mainly responsible for its replication defect. Protein C7 interacted with SAMD9, which antagonized the antiviral response of SAMD9 to ensure viral protein translation and replication of NTV in non-permissive cell lines. Our finding will serve as a baseline for modification of NTV in future application.

Human papillomavirus type 16 L1E7 chimeric capsomeres have prophylactic and therapeutic efficacy against papillomavirus in mice.

Genital human papillomavirus (HPV) infection is the primary cause of cervical cancer in women. Although the HPV recombinant L1 protein was recently licensed as an available vaccine, it has numerous shortcomings. New vaccination strategies should be considered. To enable the design of a prophylactic and therapeutic low-cost vaccine candidate, chimeric HPV16 L1DeltaC34E7N1-60 capsomeres were produced in Escherichia coli. The immune characteristics and potential prophylactic and therapeutic effects of these capsomeres were examined in C57BL/6 mice. Following protein purification and renaturation, the majority of the recombinant chimeric proteins (L1DeltaC34E7N1-60) assembled into capsomeres. These capsomeres were able to induce conformational and neutralizing antibodies against HPV virus-like particles and trigger cell-mediated specific immune responses against the L1 and E7 peptides. In vivo tumor challenge assays showed that mice immunized with the capsomeres were protected against a challenge with both C3 and TC-1 tumor cells. Furthermore, in vivo tumor rejection assays showed that capsomeres have therapeutic efficacy in mice following inoculation with C3 and TC-1 tumor cells. Chimeric capsomeres are capable of preventing and eliminating HPV16 infection. Therefore, our study has provided an economical vaccine candidate.

[Highly efficient expression of codon-optimized human papillomavirus 16 L2E7 gene in Escherichia coli].

OBJECTIVE: To enhance the expression level of human papillomavirus (HPV) 16 L2E7 in Escherichia coli (E. coli), in aim of providing high-level expression of HPV16 L2E7 strain for pre-clinical high-throughout production. METHODS: The whole L2E7 gene was optimized by software of Synthetic Gene Designer, reflecting E. coli codon usage. Two parts of codon-optimized gene were cloned into pET9a vector step by step. The positive clone, which was sequenced to be corrected, was transfected to BL21 (DE3+) via isopropyl-beta-D-thiogalactoside (IPTG) induction. They produced the HPV16 L2E7 fusion protein, which was further detected by SDS-PAGE and Western blot. The induction temperature, induction time, and IPTG concentration were also optimized by a series of experiments. Further purification modes of this protein were also explored. RESULTS: Codon-optimized HPV16 L2E7 was highly expressed in E. coli. The target protein accounted for nearly 60% of the total cell extract. CONCLUSION: High-level expression of HPV16 L2E7 was successfully constructed.

[Optimized expression, preparation of human papillomavirus 16 L2E7 fusion protein and its inhibitory effect on tumor growth in mice].

HPV16 L2E7 is a fusion protein used for therapeutical vaccine targeting HPV virus. To increase its expression in Escherichia coli, we optimized the codon usage of HPV16 l2e7 gene based on its codon usage bias. The optimized gene of HPV16 sl2e7 was cloned into three different vectors: pGEX-5X-1, pQE30, ET41a, and expressed in JM109, JM109 (DE3) and BL21 (DE3) lines separately. A high expression line was selected with pET41a vector in BL21 (DE3) cells. After optimization of the growth condition, including inoculation amount, IPTG concentration, induction time and temperature, the expression level of HPV16 L2E7 was increased from less than 10% to about 28% of total protein. HPV16 L2E7 protein was then purified from 15 L culture by means of SP Sepharose Fast Flow, Q Sepharose Fast Flow and Superdex 200 pg. After renaturing, HPV16 L2E7 protein with ≥ 95% purity was achieved, which was confirmed via SDS-PAGE gel and Western blotting. The combined use of purified HPV16 L2E7 and CpG helper has shown clear inhibition of tumor growth in mice injected with tumor cells, with six out of eight mice shown no sign of tumor. This study lays a solid foundation for a new pipeline of large-scale vaccine production.

[Non-replicating recombinant vaccinia virus expressing HPV16 E6 and E7 proteins elicits anti-tumor immunity in mice].

OBJECTIVE: To investigate the anti-tumor immunity of the non-replicating recombinant vaccinia virus expressing HPV16 E6 and E7 proteins. METHODS: C57BL/6 mice were immunized by non-replicating recombinant vaccinia virus (NTVJmE6E7), and then specific CTLs were determined. Immune protection effects were evaluated by challenges of different doses of TC-1 tumor cells. Immunotherapeutic effects in form of recurrence were evaluated on the tumor-removed mice. RESULTS: Mice immunized by NTVJmE6E7 could generate TC-1 cell specific cytotoxic T lymphocyte (CTL). Mice boosted with NTVJmE6E7 could tolerate the challenge of 1 x 10(4) TC-1 cells. NTVJmE6E7 could effectively prevent the tumor recurrence in the tumor-removed mice. CONCLUSION: NTVJmE6E7 can be taken as a candidate of therapeutic vaccine for HPV-associated tumors and their precursor lesions.

[The high expression of HPV31 and 52 L2 fusion protein in E. coli and detection of its immunogenicity].

OBJECTIVE: To express HPV31 and 52 L2 fusion protein and detect its immunogenicity. METHODS: According to the amino acid sequences of HPV31 and 52 L2 11-200AA published in the GenBank database, weartificially synthesized the HPV31 and 52 L2 fusion gene which was optimized according to Escherichia coli codon usage and encodes 11-200 amino acid of HPV31 and HPV52 L2, then cloned it into pET-9a vector. The HPV31 and 52 L2 fusion protein was expressed in Prokaryotic expression system and the mice were immunized with the fusion protein after purification. The immunogenicity was characterized in vaccinated mice. RESULTS: HPV31 and 52 L2 fusion protein was highly expressed in E. coli, the amount of fusion protein is nearly 20% of the total bacterial protein. The purified fusion protein with aluminum adjuvant could induce specific high titer of IgG antibodies detected by ELISA, and also induce the neutralizing antibodies against pseudovirus of HPV31 and HPV52 and cross-neutralizing antibodies against pseudovirus of HPV45, 58, 16, 18. CONCLUSION: HPV31 and 52 L2 fusion protein could induce neutralizing and cross-neutralizing antibodies against HPV pseudovirus. It provides laboratory basis for development of HPV L2 protein vaccine.

[The expression and preliminary evaluation of HPV6bL2deltaN360E7E6 fusion protein in E. coli for genital warts].

OBJECTIVE: To express HPV6bL2deltaN360E7E6 fusion protein in E. coli and preliminarily evaluate its immune effect. METHODS: Three HPV6b gene fragments, which were L2(1-360 bp), E7 and E6, were fused by overlapping PCR, then were inserted into a prokaryotic expression vector and expressed in E. coli. C57BL/6 mice were immunized with purified fusion protein plus Al (OH)3 and/or CpG adjuvants through intramuscular route, the cellular and humoral immune responses were detected by IFN-gamma ELISPOT and ELISA respectively. RESULTS: Protein plus CpG adjuvant could induce the strongest cellular immune response to E7 and E6, high antibody titer against L2 could be detected in all immunized groups but there were no significant difference among these groups. CONCLUSIONS: HPV6bL2deltaN360E7E6 gene was successfully cloned into pQE30 vector and expressed in E. coli, the fusion protein was also purified and proved that could induce strong cellular and humoral immune responses with appropriate adjuvant in C57BL/ 6 mice and could be used for future research.

[Identification specific T lymphocyte epitopes on E6 protein of human papillomavirus type 18 in mice].

OBJECTIVE: To identify specific T lymphocyte epitope on E6 protein of human papillomavirus type 18 in mice. METHODS: Infection with one recombinant vaccinia virus rVVJ18 E7, E6 respectively in C57 BL/6 and BALB/c mice, specific cellular immune responses were detected by ELISPOT or intracellular cytokine stainings by using a series of overlapping synthetic peptides covering full length of the amino acid sequence of E6 and E7 proteins or various truncated peptides. RESULTS: The rVVJ18 E7, E6 generated significant E6 specific T-cell immune responses in vaccinated mice. Mapping of the epitope of E6 revealed that the peptides E6(67-75 ( KCIDFYSRI) and E6(60-68) (IPHAAGHKC) presented respectively by C57BL/6 and BALB/c mice were the optimal peptides to activate E6-specific CD8+ T lymphocytes. However no positive cellular immune responses stimulated with various E7 peptides were detected by ELISPOT in immunized mice. CONCLUSIONS: Two specific T lymphocyte epitopes were identified on E6 protein in C57BL/6 and BALB/c mice, which will provide the basis to evaluate cellular immune response elicited by HPV18 E6 protein based vaccine.

[Construction of recombinant vaccinia virus expressing HPV18E7E6 fusion proteins and detection of its immunogenicity in mice].

OBJECTIVE: To construct one recombinant vaccinia virus expressing the HPV18 E6 and E7 fusion proteins as HPV18 therapeutic vaccine candidate, and test its immunogenicity. METHODS: The fusion E7E6 genes were synthesized and mutated to inactivate their oncogenic potential, and inserted into a vaccinia virus plasmid vector to construct one recombinant vaccinia virus. Finally its immunogenicity was characterized in immunized mice. RESULTS: One recombinant vaccinia virus expressing HPV18 E7E6 fusion proteins was constructed. Sequencing results of PCR products and Western blot tests showed that the E7E6 fusion genes were correct and expressed in CEF cells infected with the recombinant vaccinia virus. The specific antibodies against E6 and E7 proteins were elicited, however no positive responses were detected by ELISPOT in immunized mice. CONCLUSIONS: One recombinant vaccinia virus expressing HPV18 E7E6 fusion proteins was generated and elicited specific antibodies against E6 and E7 proteins, but detected no positive cellular immune responses in immunized mice, which will provide the basis to develop the different animal model for examining the cellular immune responses of HPV18E6 and E7 proteins.

[Prokaryotic expression and purification of human papillomavirus type 11 L2E7 fusion protein vaccine and its immunnogenicity].

OBJECTIVE: To construct the Escherichia coli (E. coli) prokaryotic expression system pET9aHPV11L2E7, purify the fusion protein L2E7 and study the immunnogenicity of the protein. METHODS: The HPV11 L2, E7 coding region was amplified from condyloma acuminata tissue specimen by PCR. The recombinant plazmid pET9aHPV11L2E7 was established and sequenced. Fusion protein L2E7 (553 amino acids) was expressed in host strain BL21 (DE3plus) by IPTG inducing and identified by using SDS-PAGE and Western blotting. Then L2E7 protein purified with CM column was inoculated to Balb/c mice and its cell-mediated and humoral immunnogenicity was assessed by IFN-gamma enzyme-linked immunospot (ELISPOT) and enzyme-linked immunosorbent assay (ELISA). RESULTS: The E. coli prokaryotic expression system pET9aHPV11L2E7 was established and the purified fusion protein L2E7 was obtained successfully. The mice in vivo experiment indicated that the purified protein L2E7 could induce HPV11E7 specific cell-mediated immune responses and high level HPV L2E7 antibody was detected in serum. CONCLUSION: The purified fusion protein L2E7 could induce specific cell-mediated and humoral immune responses. It can be used as a candidate of genital wart immune therapeutic vaccine.

[Expression of the human papillomavirus type 16L/E7 fusion protein in E. coli and observation of its immunogenicity in mice].

BACKGROUND: Many epidemiological and experimental evidences prove that cervical cancers are strongly associated with genital high-risk types of human papillomavirus (HPV). HPV16 is present in 50% of the tumor specimens. Thus, it is important to develop vaccines against HPV16 and cervical cancer. The authors studied the expression of the HPV16 L1DeltaCE7N fusion protein in E. coli and observed its immunogenicity. METHODS: The fragment of HPV16 L1DeltaC gene and the E7N gene were amplified by PCR separately; the fusion gene named L1DeltaCE7N was generated by fusing E7N to the C terminal of L1DeltaC then the chimeric gene was cloned into prokaryotic expression vector pGEX-2T and expressed in E. coli strain JM109. The L1DeltaCE7N protein expressed were detected by Western blot. Finally its immunogenicity was characterized in immunized mice. RESULTS: It was proved that the sequence and open reading frame of fusion gene L1DeltaE7N was correct by sequencing; SDA-PAGE gel analysis showed that HPV16 L1/E7 fusion protein was highly expressed in E. coli; the protein was expressed as soluble form and the molecular weight was about 85 x 10(3). The fusion protein could be purified by affinity chromatography and gel filtration. The ELISA result indicated that L1/E7 could elicit specific antibodies against L1 and E7 in immunized mice. In vivo tumor protection test indicated that tumor formation was retarded or prevented in the mice after vaccination with L1/E7, when C57 BL/6 mice were challenged by syngeneic HVP16E6 and E7 transformed tumor cells. CONCLUSION: HPV16L1/E7 fusion protein was expressed in E. coli, it can be a candidate for prophylactic and therapeutic vaccine for HPV16-associated infection and tumors.

[Construction of recombinant vaccina virus of HPV16 and its antitumor effect].

BACKGROUND & OBJECTIVE: Cervical cancer is tightly related with high-risk types of human papillomavirus (HPV), among which HPV16 is most common. This study was to construct HPV16 L1, L2, E67 coexpression non-replication vaccina virus, and explore its immunization effects. METHODS: HPV16 major capsid protein L1/L2 genes and HPV16 early E6/E7 genes were inserted into a vaccina virus expression vector to construct recombinant vaccina virus NTVJE67CKL1L2 by homologous recombination; the recombinant virus was identified by DNA hybridization and Western blot. C57BL/6 mice were immunized by NTVJE67CKL1L2; specific antibodies and specific cytotoxic T lymphocytes (CTLs) were detected. Immune protective effects of NTVJE67CKL1L2 on the mice were evaluated by challenges of TC-1 tumor cells. RESULTS: HPV16 L1/L2/E6/E7 genes were integrated into vaccina virus DNA; Western blot confirmed that full-length L1/L2/E67 proteins were co-expressed in chicken embryo fibroblast (CEF) cells infected with the recombinant virus. NTVJE67CKL1L2 elicited specific antibodies against HPV16 L1/L2/E6/E7 and generated TC-1 cell-specific CTLs in mice. Mice boosted with NTVJE67CKL1L2 could tolerate the challenge of 1x10(4) TC-1 cells. CONCLUSION: NTVJE67CKL1L2 can be taken as a candidate of prophylactic and therapeutic vaccine for HPV-associated tumors and their precursor lesions.

[Construction and identification of non-replication recombinant vaccinia virus co-expressing human papillomavirus type 16 L1/L2/E6/E7 proteins].

OBJECTIVE: To generate a human papillomavirus (HPV16) prophylactic and therapeutic vaccine candidate for cervical cancer. METHODS: HPV16 major capsid protein L1 gene/minor capsid protein L2 gene and HPV16 early E6/E7 genes were inserted into a vaccinia virus expression vector. A strain of non-recombinant vaccinia virus containing the sequences was obtained through a homologous recombination and identified. RESULTS: DNA hybridization confirmed that the HPV16L1/L2/E6/E7 genes were integrated into vaccinia virus DNA. Western Blot result showed that full-length L1/L2/E6/E7 proteins were co-expressed in CEF cells infected with the recombinant virus. CONCLUSION: NTVJE6E7CKL1L2 could be taken as a candidate of prophylactic and therapeutic vaccine for HPV-associated tumors and their precancerous transformations.

[Construction of recombinant vaccinia virus co-expressing mutant E6 plus E7 proteins and detection of its immunogenicity and antitumor response].

OBJECTIVE: To generate a candidate HPV16 vaccine for experimental and therapeutical use for cervical cancer. METHODS: The mutants of HPV16 early E6 and E7 genes were inserted into a vaccinia virus expression vector. A strain of recombinant vaccinia virus was constructed through homologous recombination. RESULTS: Showed that the mutant E6 and E7 genes were located at TK gene region of vaccinia virus Tiantan strain in a head to head orientation under the control of early/late promoters, H6 and 7.5K respectively. Studies in mice indicated that VmE6E7 could elicit specific antibodies against E6 and E7, and retarded or prevented tumor development in a proportion of C57 BL/6 mice challenged by syngeneic HPV16E6 and E7 transformed tumor cells. CONCLUSIONS: The success in constructing VmE6E7 provides a basis for the further development of HPV16 therapeutic vaccine.

[Modification of HPV type 16 E6 and E7 genes, and analysis of stability and immunogenicity of the modified proteins].

BACKGROUND: To select the mutants of HPV type 16 E6 and E7 genes suitable for construction of vaccine for treatment of cervical cancer. METHODS: E6 and E7 genes were modified by site-directed mutagenesis. Several recombinant vaccina viruses were constructed by inserting the E6 or E7 mutants into the genome of vaccina virus Tiantan strain and employed to study their antigenicity. RESULTS: Western blot assay showed that the E6 ?mutant? with substitution of Gly for Leu at amino acid site 50 and E7 mutant with substitution of Gly for Cys-24 and Glu-26 had no effect on their stability and antigenicity, but change of the Cys at position 91 of E7 dramatically reduced its stability and antigencity. Conclusion The results confirmed that the Zinc-finger structure at the E7 C-terminal? plays an important role in the integrity and stability of E7 protein.