B5R Gene Research Articles

Redirecting anti-Vaccinia virus T cell immunity for cancer treatment by AAV-mediated delivery of the VV B8R gene

Immunotherapies, such as immune checkpoint inhibitors (ICIs) and chimeric antigen receptor-T (CAR-T) cells, are only efficient in a small proportion of tumor patients. One of the major reasons for this is the lack of immune cell infiltration and activation in the tumor microenvironment (TME). Recent research reported that abundant bystander CD8+ T cells targeting viral antigens exist in tumor infiltrates and that virus-specific memory T cells could be recalled to kill tumor cells. Therefore, virus-specific memory T cells may be effective candidates for tumor immunotherapy. In this study, we established subcutaneous tumor mice models that were pre-immunized with Vaccinia virus (VV) and confirmed that tumor cells with ectopic expression of the viral B8R protein could be recognized and killed by memory T cells. To create a therapeutic delivery system, we designed a recombinant adeno-associated virus (rAAV) with a modified tumor-specific promoter and used it to deliver VV B8R to tumor cells. We observed that rAAV gene therapy can retard tumor growth in VV pre-immunized mice. In summary, our study demonstrates that rAAV containing a tumor-specific promoter to restrict VV B8R gene expression to tumor cells is a potential therapeutic agent for cancer treatment in VV pre-immunized or VV-treated mice bearing tumors.

Characterization of a novel nicotinamide adenine dinucleotide-cytochrome b5 reductase mutation associated with canine hereditary methemoglobinemia.

Hereditary methemoglobinemia associated with nicotinamide adenine dinucleotide-cytochrome b5 reductase (b5R) deficiency is a rare autosomal recessive disorder in animals. Recently, nonsynonymous b5R gene (CYB5R3) variants have been reported to be associated with canine and feline hereditary methemoglobinemia. However, the underlying molecular mechanisms of canine and feline methemoglobinemia caused by these nonsynonymous variants have not yet been reported. Previously, we reported a Pomeranian dog family with hereditary methemoglobinemia, carrying CYB5R3 mutation of an A>C transition at codon 194 in exon 7, replacing an isoleucine residue with leucine (p.Ile194Leu). In this study, we investigated the enzymatic and structural properties of the soluble form of wild-type and Ile194Leu canine b5Rs to characterize the effects of this missense mutation. Our results showed that the kinetic properties of the mutant enzyme were not affected by this amino acid substitution. The secondary structure of the wild-type and Ile194Leu b5Rs detected by circular dichroism showed a similar pattern. However, the mutant enzyme exhibited decreased heat stability and increased susceptibility to trypsin hydrolysis. Moreover, the thermostability and unfolding measurements indicated that the mutant enzyme was more sensitive to temperature-dependent denaturation than the wild-type b5R. We concluded from these results that unstable mutant enzyme properties with normal enzymatic activity would be associated with hereditary methemoglobinemia in the Pomeranian dog family.

Evaluation of Oncolytic Viral Therapy Combined with or without Artificial Immunoadjuvant Cell Therapy in Immunocompetent Mouse Tumor Models

Oncolytic viruses have a great potential to provide clinical improvement for cancer patients who do not respond to conventional therapies. The mechanisms of action include direct infection, immune-mediated cell death, and vascular collapse. We previously reported that a miRNA-regulated, thymidine kinase-deleted vaccinia virus (MDVV) specifically infects multiple myeloma (MM) cells and showed that the intravenous injection of MDVV significantly prolonged survival of severe combined immunodeficiency mice harboring a subcutaneous human RPMI8226 MM tumor (Blood 2014:124:2082, Mol Ther Oncolytics 2017;6:57). Although it was highly effective, there remained to be confirmed whether the MDVV would be similarly effective in the immunocompetent setting. To address this question, we performed several in vivo infection using immunocompetent C57BL/6 (B6) models.A vaccinia virus (VV) strain LC16, which was derived from the Lister strain, was widely used in 1970s for the smallpox eradication program with no severe adverse effect. Because its safety is robust, we used the LC16 for the backbone of the MDVV. To improve its safety and tumor specificity, we deleted viral thymidine kinase gene, and introduced miRNA (let-7a)-target sequences into the critical viral B5R gene. Because let-7a is abundant in normal differentiated tissues, and the endogenous let-7a down-regulates viral B5R gene, the MDVV becomes far more tumor specific (Mol Ther Oncolytics 2017;6:57).To observe whether the MDVV protects immunocompetent mice from MM, we generated a mouse MM model by transplanting 1x10^6 Vk*Myc tumor cells into syngeneic B6 mice. After confirming M-proteins in the serum, 1x10^7 pfu of MDVV was injected intravenously. Contrary to our expectation, we could not observe a clearly infected region, nor a prolongation of survival of these mice; probably due to difference in infectivity between species. Although mouse derived cell lines are generally insensitive to vaccinia virus as compared with human cell lines, a mouse lung carcinoma cell line 3LL showed a relatively high infectivity to VV in our screening in vitro infection experiment (Figure), therefore we used the B6/3LL subcutaneous tumor model instead of B6/Vk*Myc model.The B6/3LL mice were injected with mock or 1x10^7 pfu MDVV intravenously when the subcutaneous tumor became more than 5 mm in diameter. Although viral signal (determined by in vivo imaging using luciferase) disappeared 3 days after infection, the therapeutic benefit had remained, and prolongation of survival was observed Because natural killer (NK) cells were thought to play in the clearance of VV in the B6 mice, we attempted to extend the duration of infection by depleting NK cells using anti-asialo GM1 antibody, which, we thought, enhances viral oncolytic activity. As expected, we observed a persistent viral signal for up to 2 weeks in anti-asialo GM1 antibody treated B6/3LL mice. However, the anti-tumor effect seen with anti-asialo GM1 antibody was rather milder than that of without anti-asialo GM1 antibody, indicating the importance of NK activities in anti-tumor immunity.Based on this finding, we, in reverse, tried to stimulate the anti-tumor immunity using artificial immunoadjuvant cells co-expressing CD1d and tumor antigen/GM-CSF fusion protein. The CD1d is one of non-classical MHC molecules and is involved in the presentation of lipid antigens. When α-GlaCer is loaded on the CD1d, it activates natural killer T (NKT) cells through the interaction with invariant T cell receptor on NKT cells. When activated, NKT cells produce Th1 and Th2 cytokines, and activate NK cells. GM-CSF stimulates antigen-presenting cells (APCs), and fusion proteins of tumor antigen and GM-CSF are more efficiently taken up by APCs than tumor antigen alone (Nature 1993, 362:755). We generated the artificial immunoadjuvant cells by transducing 3LL tumor cells with lentiviral vectors expressing CD1d and ovalbumin (OVA)/GM-CSF fusion protein. After loading with α-GlaCer, the immunoadjuvant cells were irradiated with 50Gy, and injected intravenously into B6/3LL-OVA mice. We observed a significant reduction in tumor mass and prolongation of survival as compared with mock-injected mice. Currently, we are exploring the optimal setting of combination with MDVV virotherapy and immunoadjuvant cell therapy. In this meeting, we will present these results. DisclosuresNo relevant conflicts of interest to declare.

Protection of mice against the highly pathogenic VVIHD-J by DNA and fowlpox recombinant vaccines, administered by electroporation and intranasal routes, correlates with serum neutralizing activity

The control of smallpox was achieved using live vaccinia virus (VV) vaccine, which successfully eradicated the disease worldwide. As the variola virus no longer exists as a natural infection agent, mass vaccination was discontinued after 1980. However, emergence of smallpox outbreaks caused by accidental or deliberate release of variola virus has stimulated new research for second-generation vaccine development based on attenuated VV strains. Considering the closely related animal poxviruses that also arise as zoonoses, and the increasing number of unvaccinated or immunocompromised people, a safer and more effective vaccine is still required. With this aim, new vectors based on avian poxviruses that cannot replicate in mammals should improve the safety of conventional vaccines, and protect from zoonotic orthopoxvirus diseases, such as cowpox and monkeypox. In this study, DNA and fowlpox (FP) recombinants that expressed the VV L1R, A27L, A33R, and B5R genes were generated (4DNAmix, 4FPmix, respectively) and tested in mice using novel administration routes. Mice were primed with 4DNAmix by electroporation, and boosted with 4FPmix applied intranasally. The lethal VVIHD-J strain was then administered by intranasal challenge. All of the mice receiving 4DNAmix followed by 4FPmix, and 20% of the mice immunized only with 4FPmix, were protected. The induction of specific humoral and cellular immune responses directly correlated with this protection. In particular, higher anti-A27 antibodies and IFNγ-producing T lymphocytes were measured in the blood and spleen of the protected mice, as compared to controls. VVIHD-J neutralizing antibodies in sera from the protected mice suggest that the prime/boost vaccination regimen with 4DNAmix plus 4FPmix may be an effective and safe mode to induce protection against smallpox and poxvirus zoonotic infections. The electroporation/intranasal administration routes contributed to effective immune responses and mouse survival.

Vaccinia virus strain LC16m8 defective in the B5R gene keeps strong protection comparable to its parental strain Lister in immunodeficient mice

BackgroundAttenuated vaccinia virus strain, LC16m8, defective in the B5R envelope protein gene, is used as a stockpile smallpox vaccine strain in Japan against bioterrorism: the defect in the B5R gene mainly contributes to its highly attenuated properties. MethodsThe protective activity of LC16m8 vaccine against challenge with a lethal dose of vaccinia Western Reserve strain was assessed in wild-type and immunodeficient mice lacking CD4, MHC class I, MHC class II or MHC class I and II antigens. ResultsThe immunization with LC16m8 induced strong protective activity comparable to that of its parent strain, Lister (Elstree) strain, in wild-type mice from 2 days to 1 year after vaccination, as well as in immunodeficient mice at 2 or 3 weeks after vaccination. These results implicated that the defect in the B5R gene hardly affected the potential activity of LC16m8 to induce innate, cell-mediated and humoral immunity, and that LC16m8 could be effective in immunodeficient patients. ConclusionLC16m8 with truncated B5 protein has an activity to induce immunity, such as innate immunity and subsequent cell-mediated and humoral immunity almost completely comparable to the activity of its parental strain Lister.

Isolation of buffalo poxvirus from clinical case and variations in the genetics of the B5R gene over fifty passages.

Outbreaks of buffalopox affect udder and teats, which may ultimately lead to mastitis in dairy buffalo and can significantly compromise the production. In this study, we report isolation of buffalo poxvirus and sequence analysis of the B5R gene collected from the buffalo clinically suspected to be poxvirus infected. The virus was isolated on BHK-21 cell line and was passaged for 50 times, B5R gene was amplified and sequenced using gene-specific primers, and analyzed at both nucleotide and amino acid levels. Phylogenetically, the isolate can be classified close to the previously reported Pakistani and Indian isolates with certain level of differential clustering patterns. Three significant putative mutations (I2K, N64D, and K111E) were observed in the B5R protein. The K111E was common with previous human isolate from Karachi, Pakistan in 2005. These mutations differed from poxviruses reported from the neighboring countries. Some deletion mutations were observed which were recovered in upcoming passages. The K111E mutation suggests potential to cause zoonotic infection in human all over the country.

Targeting DNA vaccines to dendritic cells using a small peptide to enhance immune response (VAC4P.1110)

Abstract Dendritic cell targeting of antigens greatly enhances immunogenicity. Although there are several reagents (DEC205, Dectin-1, Langerin, Clec9, nanoparticle, etc) to target protein antigens to DCs, there is still no method to target DNA vaccines directly to DCs. Here we show that a small peptide derived from the rabies virus glycoprotein, fused to protamine residues (RVG-P) can target DNA to DCs resulting in enhanced T-cell and humoral responses. Targeted delivery of DNA vaccine encoding the immunodominant Vaccinia virus B8R gene to DCs with RVG-P was able to re-stimulate Vaccinia-specific memory T cells in vitro. Moreover, a single iv injection of B8R gene bound to RVG-P without any adjuvants was able prime a Vaccinia-specific T-cell response that was able to rapidly clear a subsequent Vaccinia virus challenge in mice. Finally, immunization of mice with a DNA vaccine encoding with West Nile virus (WNV) prM and E proteins via RVG-P elicited high titers of WN neutralizing antibodies that protected mice from lethal WNV challenge. Thus, RVG-P provides a tool to target DNA vaccines to DCs to elicit robust T-cell and humoral immune responses.

Thymidine Kinase-Deleted, let7a-Regulated Vaccinia Virus Specifically Infects and Lyses Myeloma Cells in a Mouse Myeloma Model

Background: Vaccinia virus is a tool for a novel cancer therapy that infects and lyses cancer cells. Clinical trials targeting solid tumors are currently ongoing, and promising results are reported. Although in vitro studies suggest that hematological malignancies have only modest susceptibility in general, we found that multiple myeloma shows an exceptionally high susceptibility to vaccinia virus, therefore, we focused on myeloma. Because tumor specific infection is the key for a successful oncolytic therapy, we modified thymidine kinase (TK) gene and B5R gene in the vaccinia genome to make it more tumor-specific. Efficacy and safety was determined by the in vitro and in vivo mouse infection models.Methods: Vaccinia TK is required for viral replication, but it is dispensable in tumor cells because high level of TK produced by the tumor can substitute it. In order to delete TK gene, we generated a plasmid vector which have a homologous sequence of TK (pTK), and the firefly luciferase (Fluc) gene with the PSFL110 promoter was inserted into the middle of TK gene. By the co-transfection of 143B cells with the pTK-Fluc and parental vaccinia virus LC16mO (derived from Lister clone 16), a homologous recombination occurred and the TK-deleted vaccinia virus was generated. Vaccinia B5R gene is essential for the viral transmission from infected cells to adjacent cells. To regulate B5R gene expression and inhibit viral transmission in normal cells, we inserted a let-7a target sequence into the downstream of B5R in the pB5R plasmid. Let-7a is a micro RNA of which expression is lost in tumor cells but maintained in most of the normal cells, therefore, we hypothesized that endogenous let-7a in normal cells will inhibit B5R, inhibit viral transmission, and improve safety. By the co-transfection of RK13 cells with the pB5Rgfp-let7a and TK-deleted vaccinia virus, a homologous recombination occurred, and TK-deleted let-7a-regulated vaccinia virus was generated.Results: To find out a good target for this therapy, we first infected different cell lines including Kasumi-1, NB4, NALM6, Jurkat, OM9;22, K562 (these are from leukemia), FL218, Daudi (lymphoma), MT2, TLO-m1 (ATL), and RPMI8226, U266, NCI-H929 (myeloma) with a vaccinia virus. Very interestingly, myeloma-derived cell lines showed an exceptionally high susceptibility (10-100 times higher than others), and this susceptibility was as high as solid tumors; therefore we chose multiple myeloma as the target. To confirm that regulation of B5R by the let-7a improves myeloma-specific infection, we infected a myeloma cell line RPMI8226 as well as normal skin fibroblasts with the recombinant B5Rgfp-let7a vaccinia virus. In vitro infection of a RPMI8226 cells showed a significant infectivity even with a very low titer (MOI=0.1), while control normal cells (human skin fibroblasts) were not infected at all. Without the let7a-regulation, the vaccinia virus infected both myeloma cells and normal cells at the same titer, suggesting that let-7a-mediated regulation clearly works. As an in vivo infection model, 1x107 RPM8226-Rluc cells were injected into immunodeficient mice (CB.17-SCID) subcutaneously. Four weeks later, 1x106 pfu of virus (Parent; TK-deleted; TK and let7a double regulated) was administered via an intravenous injection, and tumor volume and the amount of virus were determined weekly by the in vivo imaging system with renilla and firefly luciferases. Without any regulation, vaccinia virus infected not only myeloma cells but also normal tissues, and mice developed pock lesions in the ear, nose, mouth, foot, and tail, resulting in death within 21 to 24 days. TK-deletion significantly alleviated viral toxicity but still caused death 42 days after infection. In contrast, infection with TK and let-7a double regulated vaccinia virus was clearly limited in myeloma, and all mice remained alive and the size of myeloma shrunk continuously.Conclusions: These data suggest that the TK-let7a-double regulated vaccinia virus infects and kills myeloma cells specifically, and will be a good candidate for the future clinical application. DisclosuresNo relevant conflicts of interest to declare.

Targeting DNA vaccines to myeloid cells using a small peptide.

Targeting DNA vaccines to dendritic cells (DCs) greatly enhances immunity. Although several approaches have been used to target protein Ags to DCs, currently there is no method that targets DNA vaccines directly to DCs. Here, we show that a small peptide derived from the rabies virus glycoprotein fused to protamine residues (RVG-P) can target DNA to myeloid cells, including DCs, which results in enhanced humoral and T-cell responses. DCs targeted with a DNA vaccine encoding the immunodominant vaccinia B8R gene via RVG-P were able to restimulate vaccinia-specific memory T cells in vitro. Importantly, a single i.v. injection of B8R gene bound to RVG-P was able to prime a vaccinia-specific T-cell response that was able to rapidly clear a subsequent vaccinia challenge in mice. Moreover, delivery of DNA in DCs was enough to induce DC maturation and efficient Ag presentation without the need for adjuvants. Finally, immunization of mice with a DNA-vaccine encoding West Nile virus (WNV) prM and E proteins via RVG-P elicited high titers of WNV-neutralizing Abs that protected mice from lethal WNV challenge. Thus, RVG-P provides a reagent to target DNA vaccines to myeloid cells and elicit robust T-cell and humoral immune responses.

Systemically administered DNA and fowlpox recombinants expressing four vaccinia virus genes although immunogenic do not protect mice against the highly pathogenic IHD-J vaccinia strain

The first-generation smallpox vaccine was based on live vaccinia virus (VV) and it successfully eradicated the disease worldwide. Therefore, it was not administered any more after 1980, as smallpox no longer existed as a natural infection. However, emerging threats by terrorist organisations has prompted new programmes for second-generation vaccine development based on attenuated VV strains, which have been shown to cause rare but serious adverse events in immunocompromised patients. Considering the closely related animal poxviruses that might also be used as bioweapons, and the increasing number of unvaccinated young people and AIDS-affected immunocompromised subjects, a safer and more effective smallpox vaccine is still required. New avipoxvirus-based vectors should improve the safety of conventional vaccines, and protect from newly emerging zoonotic orthopoxvirus diseases and from the threat of deliberate release of variola or monkeypox virus in a bioterrorist attack. In this study, DNA and fowlpox recombinants expressing the L1R, A27L, A33R and B5R genes were constructed and evaluated in a pre-clinical trial in mouse, following six prime/boost immunisation regimens, to compare their immunogenicity and protective efficacy against a challenge with the lethal VV IHD-J strain. Although higher numbers of VV-specific IFNγ-producing T lymphocytes were observed in the protected mice, the cytotoxic T-lymphocyte response and the presence of neutralising antibodies did not always correlate with protection. In spite of previous successful results in mice, rabbits and monkeys, where SIV/HIV transgenes were expressed by the fowlpox vector, the immune response elicited by these recombinants was low, and most of the mice were not protected.

Effect of the Deletion of Genes Encoding Proteins of the Extracellular Virion Form of Vaccinia Virus on Vaccine Immunogenicity and Protective Effectiveness in the Mouse Model

Antibodies to both infectious forms of vaccinia virus, the mature virion (MV) and the enveloped virion (EV), as well as cell-mediated immune response appear to be important for protection against smallpox. EV virus particles, although more labile and less numerous than MV, are important for dissemination and spread of virus in infected hosts and thus important in virus pathogenesis. The importance of the EV A33 and B5 proteins for vaccine induced immunity and protection in a murine intranasal challenge model was evaluated by deletion of both the A33R and B5R genes in a vaccine-derived strain of vaccinia virus. Deletion of either A33R or B5R resulted in viruses with a small plaque phenotype and reduced virus yields, as reported previously, whereas deletion of both EV protein-encoding genes resulted in a virus that formed small infection foci that were detectable and quantifiable only by immunostaining and an even more dramatic decrease in total virus yield in cell culture. Deletion of B5R, either as a single gene knockout or in the double EV gene knockout virus, resulted in a loss of EV neutralizing activity, but all EV gene knockout viruses still induced a robust neutralizing activity against the vaccinia MV form of the virus. The effect of elimination of A33 and/or B5 on the protection afforded by vaccination was evaluated by intranasal challenge with a lethal dose of either vaccinia virus WR or IHD-J, a strain of vaccinia virus that produces relatively higher amounts of EV virus. The results from multiple experiments, using a range of vaccination doses and virus challenge doses, and using mortality, morbidity, and virus dissemination as endpoints, indicate that the absence of A33 and B5 have little effect on the ability of a vaccinia vaccine virus to provide protection against a lethal intranasal challenge in a mouse model.

L1R, A27L, A33R and B5R vaccinia virus genes expressed by fowlpox recombinants as putative novel orthopoxvirus vaccines

BackgroundThe traditional smallpox vaccine, administered by scarification, was discontinued in the general population from 1980, because of the absence of new smallpox cases. However, the development of an effective prophylactic vaccine against smallpox is still necessary, to protect from the threat of deliberate release of the variola virus for bioterrorism and from new zoonotic infections, and to improve the safety of the traditional vaccine. Preventive vaccination still remains the most effective control and new vectors have been developed to generate recombinant vaccines against smallpox that induce the same immunogenicity as the traditional one. As protective antibodies are mainly directed against the surface proteins of the two infectious forms of vaccinia, the intracellular mature virions and the extracellular virions, combined proteins from these viral forms can be used to better elicit a complete and protective immunity.MethodsFour novel viral recombinants were constructed based on the fowlpox genetic background, which independently express the vaccinia virus L1 and A27 proteins present on the mature virions, and the A33 and B5 proteins present on the extracellular virions. The correct expression of the transgenes was determined by RT-PCR, Western blotting, and immunofluorescence.Results and conclusionsUsing immunoprecipitation and Western blotting, the ability of the proteins expressed by the four novel FPL1R, FPA27L, FPA33R and FPB5R recombinants to be recognized by VV-specific hyperimmune mouse sera was demonstrated. By neutralisation assays, recombinant virus particles released by infected chick embryo fibroblasts were shown not be recognised by hyperimmune sera. This thus demonstrates that the L1R, A27L, A33R and B5R gene products are not inserted into the new viral progeny. Fowlpox virus replicates only in avian species, but it is permissive for entry and transgene expression in mammalian cells, while being immunologically non–cross-reactive with vaccinia virus. These recombinants might therefore represent safer and more promising immunogens that can circumvent neutralisation by vector-generated immunity in smallpox-vaccine-experienced humans.

Sequence and phylogenetic analysis of host-range (E3L, K3L, and C7L) and structural protein (B5R) genes of buffalopox virus isolates from buffalo, cattle, and human in India

Buffalopox virus (BPXV), a close variant of vaccinia virus (VACV) has emerged as a zoonotic pathogen. The host tropism of poxviruses is governed by host-range genes. Among the host-range genes: E3L, K3L, and C7L are essential for virus replication by preventing interferon resistance, whereas B5R is essential for spread of the virus and evasion from the host's immune response as in VACV. We report sequence analysis of host-range genes: E3L, K3L, C7L, and membrane protein gene (B5R) of BPXVs from buffalo, cattle, and human from recent outbreaks in India-their phylogenetic relationship with reference strain (BP4) and other Orthopoxviruses. BPXVs revealed a sequence homology with VACVs including zoonotic Brazilian VACV-like viruses. The aa sequences of E3L and K3L genes were 100% similar in buffalo, cattle, and human isolates. However, four significant point mutations (I11K; N12K and S36F in C7L gene and D249G in B5R gene) were observed specific to buffalo isolate only. This signifies that different strains of BPXV were circulated during the outbreak. The mutations in C7L and B5R could play an important role in adaptation of BPXV in human and cattle which needs further functional studies. The strain of BPXV isolated from buffalo may not be adopted in human and cow. Various point mutations were observed in the host-range genes of reference strain (BPXV-BP4) which may be due to several passages of virus in cell culture. The phylogeny constructed based on concatenated gene sequences revealed that BPXVs are not as closely related to vaccine strain (Lister and Lister-derived strain-LC16m8), as hypothesized earlier, rather they are more closely related to reference strain (BPXV-BP4) and other vaccinia and vaccinia-like viruses such as Passatempo and Aracatuba viruses. The availability of information regarding host tropism determinants would allow us to understand molecular mechanism of species tropism of poxviruses which would be useful in unveiling new strategies to control zoonotic poxviral infections.

Recovery and molecular characterization of live Camelpox virus from skin 12 months after onset of clinical signs reveals possible mechanism of virus persistence in herds

Potentially pathogenic orthopoxviruses (OPVs) persist in nature and re-emerge for reasons we do not fully understand. New information pertaining to Orthopoxvirus (OPV) persistence in nature would significantly improve surveillance and control programs. In a recent investigation of a Camelpox virus (CMLV) outbreak in Eastern Saudi Arabia, atypical minute pox-like skin lesions (AMPL) persisted on 42.9% of convalescent camels (8.8% of herd) for more than a year after the onset of clinical signs. In order to investigate whether AMPL were related to CMLV infection, AMPL homogenates were inoculated on the chorioallantoic membranes (CAM) of specific-pathogen-free (SPF) embryonating chicken eggs (ECE). Live CMLV was recovered from AMPL homogenates. The sequences of the ATIP gene of viruses isolated in the beginning of the outbreak, and one year later from AMPL were identical, and similar to the Kazakhstan isolate CMLV M-96. Virus identity was confirmed by sequence analysis of the CMLV A33R, A27L, B5R, and L1R orthologue genes. Uninfected adult camels that came in contact with animals showing AMPL became infected within two weeks. Since AMPL were easily missed by veterinarians and camel drivers, it was concluded that CMLV survival in persistent skin lesions may be a key mechanism in maintaining the virus in previously infected camel herds during inter-epizootic periods.

MicroRNA Regulation of Glycoprotein B5R in Oncolytic Vaccinia Virus Reduces Viral Pathogenicity Without Impairing Its Antitumor Efficacy

Vaccinia virus, once widely used for smallpox vaccine, has recently been engineered and used as an oncolytic virus for cancer virotherapy. Their replication has been restricted to tumors by disrupting viral genes and complementing them with products that are found specifically in tumor cells. Here, we show that microRNA (miRNA) regulation also enables tumor-specific viral replication by altering the expression of a targeted viral gene. Since the deletion of viral glycoprotein B5R not only decreases viral pathogenicity but also impairs the oncolytic activity of vaccinia virus, we used miRNA-based gene regulation to suppress B5R expression through let-7a, a miRNA that is downregulated in many tumors. The expression of B5R and the replication of miRNA-regulated vaccinia virus (MRVV) with target sequences complementary to let-7a in the 3'-untranslated region (UTR) of the B5R gene depended on the endogenous expression level of let-7a in the infected cells. Intratumoral administration of MRVV in mice with human cancer xenografts that expressed low levels of let-7a resulted in tumor-specific viral replication and significant tumor regression without side effects, which were observed in the control virus. These results demonstrate that miRNA-based gene regulation is a potentially novel and versatile platform for engineering vaccinia viruses for cancer virotherapy.

Severe mental retardation and recessive congenital methemoglobinemia in three Indian patients: Compound heterozygous for NADH‐cytochrome b5 reductase gene mutations

The diagnosis of Type II congenital methemoglobinemia has been established in three Indian patients in a single family based on persistent cyanosis and neurological manifestations with severe enzyme deficiency in erythrocytes. Clinical evaluation showed very severe encephalopathy, microcephaly, generalized dystonia, and mild cyanosis. Molecular studies demonstrated compound heterozygosity for two mutations in the reduced nicotinamide adenine dinucleotide (NADH) cytochrome b5 reductase (b5R) gene. One was a novel mutation 705G→A, which leads to the substitution of a Trp by a stop codon at residue 235 within exon 8 and the other was a previously reported missense mutation 608G→A leading to a replacement of Cys-203 (TGC) by Try (TAC) in exon 7. Although both amino acid substitutions are located in the NADH-binding domain, the whole protein structure, especially the region between the flavin adenine dinucleotide and NADH-binding domains, is disturbed. The presence of a premature stop codon results in the production of truncated b5R and should explain the severe enzyme deficiency seen in these cases.

Comparative sequence analysis of B5R gene of zoonotic buffalo pox virus isolates with other orthopoxviruses.

The present paper describes the isolation of buffalo pox virus from scab lesions and its molecular characterization through B5R gene sequencing. During our study, pustular pox lesions were observed on the teats and mammary parenchyma of cattle and buffaloes, and the disease was of significant zoonotic importance since similar lesions were produced on the hands, legs, and face of people in close contact with the affected animals. The collected scab materials were subjected for virus isolation in 9-11-day-old chicken embryos by the chorioallontoic membrane route and in the Vero cell line. The virus was confirmed by a sensitive and rapid diagnostic polymerase chain reaction using the primers that amplify "A type inclusion" gene, and further, B5R gene of the virus was sequenced and compared with the corresponding sequences of other orthopoxviruses. The results showed high sequence homology of our isolates with other orthopoxviruses.

Cytochrome b5 and NADH cytochrome b5 reductase: genotype–phenotype correlations for hydroxylamine reduction

NADH cytochrome b5 reductase (b5R) and cytochrome b5 (b5) catalyze the reduction of sulfamethoxazole hydroxylamine (SMX-HA), which can contribute to sulfonamide hypersensitivity, to the parent drug sulfamethoxazole. Variability in hydroxylamine reduction could thus play a role in adverse drug reactions. The aim of this study was to characterize variability in SMX-HA reduction in 111 human livers, and investigate its association with single nucleotide polymorphisms (SNPs) in b5 and b5R cDNA. Liver microsomes were assayed for SMX-HA reduction activity, and b5 and b5R expression was semiquantified by immunoblotting. The coding regions of the b5 (CYB5A) and b5R (CYB5R3) genes were resequenced. Hepatic SMX-HA reduction displayed a 19-fold range of individual variability (0.06-1.11 nmol/min/mg protein), and a 17-fold range in efficiency (Vmax/Km) among outliers. SMX-HA reduction was positively correlated with b5 and b5R protein content (P<0.0001, r=0.42; P=0.01, r=0.23, respectively), and expression of both proteins correlated with one another (P<0.0001; r=0.74). A novel cSNP in CYB5A (S5A) was associated with very low activity and protein expression. Two novel CYB5R3 SNPs, R59H and R297H, displayed atypical SMX-HA reduction kinetics and decreased SMX-HA reduction efficiency. These studies indicate that although novel cSNPs in CYB5A and CYB5R3 are associated with significantly altered protein expression and/or hydroxylamine reduction activities, these low-frequency cSNPs seem to only minimally impact overall observed phenotypic variability. Work is underway to characterize polymorphisms in other regions of these genes to further account for individual variability in hydroxylamine reduction.

Molecular smallpox vaccine delivered by alphavirus replicons elicits protective immunity in mice and non-human primates

Naturally occurring smallpox was eradicated as a result of successful vaccination campaigns during the 1960s and 1970s. Because of its highly contagious nature and high mortality rate, smallpox has significant potential as a biological weapon. Unfortunately, the current vaccine for orthopoxviruses is contraindicated for large portions of the population. Thus, there is a need for new, safe, and effective orthopoxvirus vaccines. Alphavirus replicon vectors, derived from strains of Venezuelan equine encephalitis virus, are being used to develop alternatives to the current smallpox vaccine. Here, we demonstrated that virus-like replicon particles (VRPs) expressing the vaccinia virus A33R, B5R, A27L, and L1R genes elicited protective immunity in mice comparable to vaccination with live-vaccinia virus. Furthermore, cynomolgus macaques vaccinated with a combination of the four poxvirus VRPs (4pox-VRP) developed antibody responses to each antigen. These antibody responses were able to neutralize and inhibit the spread of both vaccinia virus and monkeypox virus. Macaques vaccinated with 4pox-VRP, flu HA VRP (negative control), or live-vaccinia virus (positive control) were challenged intravenously with 5 × 10 6 pfu of monkeypox virus 1 month after the second VRP vaccination. Four of the six negative control animals succumbed to monkeypox and the remaining two animals demonstrated either severe or grave disease. Importantly, all 10 macaques vaccinated with the 4pox-VRP vaccine survived without developing severe disease. These findings revealed that a single-boost VRP smallpox vaccine shows promise as a safe alternative to the currently licensed live-vaccinia virus smallpox vaccine.

The Vaccinia Virus B5 Protein Requires A34 for Efficient Intracellular Trafficking from the Endoplasmic Reticulum to the Site of Wrapping and Incorporation into Progeny Virions

The glycoproteins encoded by the vaccinia virus A34R and B5R genes are involved in intracellular envelope virus formation and are highly conserved among orthopoxviruses. A recombinant virus that has the A34R gene deleted and the B5R gene replaced with a B5R gene fused to the enhanced green fluorescent protein (B5R-GFP) gene was created (vB5R-GFP/DeltaA34R) to investigate the role of A34 during virion morphogenesis. Cells infected with vB5R-GFP/DeltaA34R displayed GFP fluorescence throughout the cytoplasm, which differed markedly from that seen in cells infected with a normal B5R-GFP-expressing virus (vB5R-GFP). Immunofluorescence and subcellular fractionation demonstrated that B5-GFP localizes with the endoplasmic reticulum in the absence of A34. Expression of either full-length A34 or a construct consisting of the lumenal and transmembrane domains restored normal trafficking of B5-GFP to the site of wrapping in the juxtanuclear region. Coimmunoprecipitation studies confirmed that B5 and A34 interact through their luminal domains, and further analysis revealed that in the absence of A34, B5 is not efficiently incorporated into virions released from the cell.