Exploitation of bacterial N-linked glycosylation to develop a novel recombinant glycoconjugate vaccine against Francisella tularensis

Glycoconjugate-based vaccines have proved to be effective at producing long-lasting protection against numerous pathogens. Here, we describe the application of bacterial protein glycan coupling technology (PGCT) to generate a novel recombinant glycoconjugate vaccine. We demonstrate the conjugation of the Francisella tularensis O-antigen to the Pseudomonas aeruginosa carrier protein exotoxin A using the Campylobacter jejuni PglB oligosaccharyltransferase. The resultant recombinant F. tularensis glycoconjugate vaccine is expressed in Escherichia coli where yields of 3 mg l⁻¹ of culture were routinely produced in a single-step purification process. Vaccination of BALB/c mice with the purified glycoconjugate boosted IgG levels and significantly increased the time to death upon subsequent challenge with F. tularensis subsp. holarctica. PGCT allows different polysaccharide and protein combinations to be produced recombinantly and could be easily applicable for the production of diverse glycoconjugate vaccines.     

Harnessing galactose oxidase in the development of a chemoenzymatic platform for glycoconjugate vaccine design

In the preparation of commercial conjugate vaccines, capsular polysaccharides (CPSs) must undergo chemical modification to generate the reactive groups necessary for covalent attachment to a protein carrier. One of the most common approaches employed for this derivatization is sodium periodate (NaIO₄) oxidation of vicinal diols found within CPS structures. This procedure is largely random and structurally damaging, potentially resulting in significant changes in the CPS structure and therefore its antigenicity. Additionally, periodate activation of CPS often gives rise to heterogeneous conjugate vaccine products with variable efficacy. Here, we explore the use of an alternative agent, galactose oxidase (GOase) isolated from Fusarium sp. in a chemoenzymatic approach to generate a conjugate vaccine against Streptococcus pneumoniae. Using a colorimetric assay and NMR spectroscopy, we found that GOase generated aldehyde motifs on the CPS of S. pneumoniae serotype 14 (Pn14p) in a site-specific and reversible fashion. Direct comparison of Pn14p derivatized by either GOase or NaIO₄ illustrates the functionally deleterious role chemical oxidation can have on CPS structures. Immunization with the conjugate synthesized using GOase provided a markedly improved humoral response over the traditional periodate-oxidized group. Further, functional protection was validated in vitro by measure of opsonophagocytic killing and in vivo through a lethality challenge in mice. Overall, this work introduces a strategy for glycoconjugate development that overcomes limitations previously known to play a role in the current approach of vaccine design.     

Glycoconjugate vaccines against Salmonella enterica serovars and Shigella species: existing and emerging methods for their analysis

The global spread of enteric disease, the increasingly limited options for antimicrobial treatment and the need for effective eradication programs have resulted in an increased demand for glycoconjugate enteric vaccines, made with carbohydrate-based membrane components of the pathogen, and their precise characterisation. A set of physico-chemical and immunological tests are employed for complete vaccine characterisation and to ensure their consistency, potency, safety and stability, following the relevant World Health Organization and Pharmacopoeia guidelines. Variable requirements for analytical methods are linked to conjugate structure, carrier protein nature and size and O-acetyl content of polysaccharide. We investigated a key stability-indicating method which measures the percent free saccharide of Salmonella enterica subspecies enterica serovar Typhi capsular polysaccharide, by detergent precipitation, depolymerisation and HPAEC-PAD quantitation. Together with modern computational approaches, a more precise design of glycoconjugates is possible, allowing for improvements in solubility, structural conformation and stability, and immunogenicity of antigens, which may be applicable to a broad spectrum of vaccines. More validation experiments are required to establish the most effective and suitable methods for glycoconjugate analysis to bring uniformity to the existing protocols, although the need for product-specific approaches will apply, especially for the more complex vaccines. An overview of current and emerging analytical approaches for the characterisation of vaccines against Salmonella Typhi and Shigella species is described in this paper. This study should aid the development and licensing of new glycoconjugate vaccines aimed at the prevention of enteric diseases.     

Strain Selection for Generation of O-Antigen-Based Glycoconjugate Vaccines against Invasive Nontyphoidal Salmonella Disease

Nontyphoidal Salmonellae, principally S. Typhimurium and S. Enteritidis, are a major cause of invasive bloodstream infections in sub-Saharan Africa with no vaccine currently available. Conjugation of lipopolysaccharide O-antigen to a carrier protein constitutes a promising vaccination strategy. Here we describe a rational process to select the most appropriate isolates of Salmonella as source of O-antigen for developing a bivalent glycoconjugate vaccine. We screened a library of 30 S. Typhimurium and 21 S. Enteritidis in order to identify the most suitable strains for large scale O-antigen production and generation of conjugate vaccines. Initial screening was based on growth characteristics, safety profile of the isolates, O-antigen production, and O-antigen characteristics in terms of molecular size, O-acetylation and glucosylation level and position, as determined by phenol sulfuric assay, NMR, HPLC-SEC and HPAEC-PAD. Three animal isolates for each serovar were identified and used to synthesize candidate glycoconjugate vaccines, using CRM₁₉₇ as carrier protein. The immunogenicity of these conjugates and the functional activity of the induced antibodies was investigated by ELISA, serum bactericidal assay and flow cytometry. S. Typhimurium O-antigen showed high structural diversity, including O-acetylation of rhamnose in a Malawian invasive strain generating a specific immunodominant epitope. S. Typhimurium conjugates provoked an anti-O-antigen response primarily against the O:5 determinant. O-antigen from S. Enteritidis was structurally more homogeneous than from S. Typhimurium, and no idiosyncratic antibody responses were detected for the S. Enteritidis conjugates. Of the three initially selected isolates, two S. Typhimurium (1418 and 2189) and two S. Enteritidis (502 and 618) strains generated glycoconjugates able to induce high specific antibody levels with high breadth of serovar-specific strain coverage, and were selected for use in vaccine production. The strain selection approach described is potentially applicable to the development of glycoconjugate vaccines against other bacterial pathogens.     

Increased efficiency of Campylobacter jejuni N-oligosaccharyltransferase PglB by structure-guided engineering

Conjugate vaccines belong to the most efficient preventive measures against life-threatening bacterial infections. Functional expression of N-oligosaccharyltransferase (N-OST) PglB of Campylobacter jejuni in Escherichia coli enables a simplified production of glycoconjugate vaccines in prokaryotic cells. Polysaccharide antigens of pathogenic bacteria can be covalently coupled to immunogenic acceptor proteins bearing engineered glycosylation sites. Transfer efficiency of PglB_Cj is low for certain heterologous polysaccharide substrates. In this study, we increased glycosylation rates for Salmonella enterica sv. Typhimurium LT2 O antigen (which lacks N-acetyl sugars) and Staphylococcus aureus CP5 polysaccharides by structure-guided engineering of PglB. A three-dimensional homology model of membrane-associated PglB_Cj, docked to the natural C. jejuni N-glycan attached to the acceptor peptide, was used to identify potential sugar-interacting residues as targets for mutagenesis. Saturation mutagenesis of an active site residue yielded the enhancing mutation N311V, which facilitated fivefold to 11-fold increased in vivo glycosylation rates as determined by glycoprotein-specific ELISA. Further rounds of in vitro evolution led to a triple mutant S80R-Q287P-N311V enabling a yield improvement of S. enterica LT2 glycoconjugates by a factor of 16. Our results demonstrate that bacterial N-OST can be tailored to specific polysaccharide substrates by structure-guided protein engineering.     

Preparation and immunological studies of protein conjugates of N-acylneuraminic acids

The overexpression of N-acetylneuraminic acid (Neu5Ac) is closely correlated with malignant transformations. Thus, Neu5Ac is an important target in the design of cancer vaccines. To study the influence of chemical modifications of Neu5Ac on its immunological properties, the α-allyl glycosides of five differently N-acylated neuraminic acid derivatives were prepared. Following selective ozonolysis of their allyl group to form an aldehyde functionality, they were coupled to keyhole limpet hemocyanin (KLH) via reductive amination. Resultant glycoconjugates were studied in C57BL/6 mice. The N-propionyl, N-iso-butanoyl and N-phenylacetyl derivatives of neuraminic acid provoked robust immune responses of various antibody isotypes, including IgM, IgG1, IgG2a and IgG3, whereas N-trifluoropropionylneuraminic acid and natural Neu5Ac were essentially nonimmunogenic. Moreover, the N-phenylacetyl and N-iso-butanoyl derivatives mainly induced IgG responses that are desirable for antitumor applications. These results raise the promise of formulating effective glycoconjugate cancer vaccines via derivatizing sialic acid residues of sialooligosaccharides. Published in 2004. This revised version was published online in July 2006 with corrections to the Cover Date.     

Glycoconjugate Vaccine Containing Escherichia coli O157:H7 O-Antigen Linked with Maltose-Binding Protein Elicits Humoral and Cellular Responses

Glycoconjugate is one of the most efficacious and safest vaccines against bacterial pathogens. Previous studies of glycoconjugates against pathogen E. coli O157:H7 focused more on the humoral responses they elicited. However, little was known about their cellular responses. In this study, we exploited a novel approach based on bacterial protein N-linked glycosylation system to produce glycoconjugate containing Escherichia coli O157:H7 O-antigen linked with maltose-binding protein and examined its humoral and cellular responses in BALB/c mice. The transfer of E. coli O157:H7 O-antigen to MBP was confirmed by western blot and MALDI-TOF MS. Mice injected with glycoconjugate O-Ag-MBP elicited serum bactericidal antibodies including anti-E. coli O157:H7 O-antigen IgG and IgM. Interestingly, O-Ag-MBP also stimulated the secretion of anti-E. coli O157:H7 O-antigen IgA in intestine. In addition, O-Ag-MBP stimulated cellular responses by recruiting Th1-biased CD4⁺ T cells, CD8⁺ T cells. Meanwhile, O-Ag-MBP induced the upregulation of Th1-related IFN-γ and downregulation of Th2-related IL-4, and the upregulation of IFN-γ was stimulated by MBP in a dose-dependent manner. MBP showed TLR4 agonist-like properties to activate Th1 cells as carrier protein of O-Ag-MBP. Thus, glycoconjugate vaccine E. coli O157:H7-specific O-Ag-MBP produced by bacterial protein N-linked glycosylation system was able to elicit both humoral and Th1-biased cellular responses.     

病原细菌多糖疫苗和多糖结合疫苗研究进展

在许多病原细菌中,荚膜多糖和O抗原多糖能够刺激机体产生保护性抗体,因此利用病原细菌的多糖制成的疫苗能有效预防传染病,同时避免了病原细菌耐药性的出现。此类疫苗包括多糖疫苗和多糖结合疫苗。细菌体内糖基化的发现,使得利用生物法生产多糖结合疫苗成了多糖结合疫苗生产的热门方向。我们简要综述了多糖疫苗和多糖结合疫苗在研究和应用方面的主要进展。     

Molecular modeling provides insights into the loading of sialic acid‐containing antigens onto CRM197: the role of chain flexibility in conjugation efficiency and glycoconjugate architecture

Vaccination is the most cost-effective way to control disease caused by encapsulated bacteria; the capsular polysaccharide (CPS) is the primary virulence factor and vaccine target. Neisseria meningitidis (Nm) serogroups B, C, Y and W all contain sialic acid, a common surface feature of human pathogens. Two protein-based vaccines against serogroup B infection are available for human use while four tetravalent conjugate vaccines including serogroups C, W and Y have been licensed. The tetravalent Menveo® conjugate vaccine is well-defined: a simple monomeric structure of oligosaccharides terminally conjugated to amino groups of the carrier protein CRM₁₉₇. However, not only is there a surprisingly low limit for antigen chain attachment to CRM₁₉₇, but different serogroup saccharides have consistently different CRM₁₉₇ loading, the reasons for which are unclear. Understanding this phenomenon is important for the long-term goal of controlling conjugation to prepare conjugate vaccines of optimal immunogenicity. Here we use molecular modeling to explore whether antigen flexibility can explain the varying antigen loading of the conjugates. Because flexibility is difficult to separate from other structural factors, we focus on sialic-acid containing CPS present in current glycoconjugate vaccines: serogroups NmC, NmW and NmY. Our simulations reveal a correlation between Nm antigen flexibility (NmW?>?NmC?>?NmY) and the number of chains attached to CRM₁₉₇, suggesting that increased flexibility enables accommodation of additional chains on the protein surface. Further, in silico models of the glycoconjugates confirm the relatively large hydrodynamic size of the saccharide chains and indicate steric constraints to further conjugation.

     

Chemoenzymatic synthesis of immunogenic meningococcal group C polysialic acid-tetanus Hc fragment glycoconjugates

Vaccination with meningococcal glycoconjugate vaccines has decreased the incidence of invasive meningitis worldwide. These vaccines contain purified capsular polysaccharides attached to a carrier protein. Because of derivatization chemistries used in the process, conjugation of polysaccharide to protein often results in heterogeneous mixtures. Well-defined vaccines are needed to determine the relationship between vaccine structure and generated immune response. Here, we describe efforts to produce well-defined vaccine candidates by chemoenzymatic synthesis. Chemically synthesized lactosides were substrates for recombinant sialyltransferase enzymes from Camplyobacter jejuni and Neisseria meningitidis serogroup C. These resulting oligosialic acids have the same α(2-9) sialic acid repeat structure as Neisseria polysaccharide capsule with the addition of a conjugatable azide aglycon. The degree of polymerization (DP) of carbohydrate products was controlled by inclusion of the inhibitor CMP-9-deoxy-NeuNAc. Polymers with estimated DP?<?47 (median DP 25) and DP?<?100 (median DP 51) were produced. The receptor binding domain of the tetanus toxin protein (TetHc) was coupled as a carrier to the enzymatically synthesized oligosialic acids. Recombinant TetHc was derivatized with an alkyne squarate. Protein modification sites were determined by trypsin proteolysis followed by LC/MS-MS^E analysis of peptides. Oligosialic acid azides were conjugated to modified TetHc via click chemistry. These chemoenzymatically prepared glycoconjugates were reactive in immunoassays with specific antibodies against either group C polysaccharide or TetHc. Sera of mice immunized with oligosialic acid-TetHc glycoconjugates contained much greater levels of polysaccharide-reactive IgG than the sera of control mice receiving unconjugated oligosialic acids. There was no apparent difference between glycoconjugates containing oligosaccharides of DP?<?47 and DP?<?100. These results suggest that chemoenzymatic synthesis may provide a viable method for making defined meningococcal vaccine candidates.     

Synthesis and immunological activity of an oligosaccharide-conjugate as a vaccine candidate against Group A Streptococcus

The synthesis and immunogenicity of a tetanus toxoid (TT)-conjugate of the hexasaccharide portion of the cell-wall polysaccharide (CWPS) of the Group A Streptococcus (GAS) is described. The synthesis relies on the reaction of an allyl glycoside of the hexasaccharide with cysteamine, followed by the reaction of the resultant amine with diethyl squarate to give the monoethyl squarate adduct. Subsequent reaction with the lysine ε-amino groups on TT gives the glycoconjugate containing 30 hexasaccharide haptens per TT molecule. The immunogenicity in mice is similar to that obtained with a native CWPS–TT conjugate, validating the glycoconjugate as a vaccine candidate against GAS infections.     

Chemical intervention in bacterial lignin degradation pathways: Development of selective inhibitors for intradiol and extradiol catechol dioxygenases

Bacterial lignin degradation could be used to generate aromatic chemicals from the renewable resource lignin, provided that the breakdown pathways can be manipulated. In this study, selective inhibitors of enzymatic steps in bacterial degradation pathways were developed and tested for their effects upon lignin degradation. Screening of a collection of hydroxamic acid metallo-oxygenase inhibitors against two catechol dioxygenase enzymes, protocatechuate 3,4-dioxygenase (3,4-PCD) and 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB), resulted in the identification of selective inhibitors D13 for 3,4-PCD (IC₅₀ 15 μM) and D3 for MhpB (IC₅₀ 110 μM). Application of D13 to Rhodococcus jostii RHA1 in minimal media containing ferulic acid led to the appearance of metabolic precursor protocatechuic acid at low concentration. Application of 1 mM disulfiram, an inhibitor of mammalian aldehyde dehydrogenase, to R. jostii RHA1, gave rise to 4-carboxymuconolactone on the β-ketoadipate pathway, whereas in Pseudomonas fluorescens Pf-5 disulfiram treatment gave rise to a metabolite found to be glycine betaine aldehyde.     

O-antigen polymerase adopts a distributive mechanism for lipopolysaccharide biosynthesis

Bacterial lipopolysaccharide (LPS) is an essential cell envelope component for gram-negative bacteria. As the most variable region of LPS, O antigens serve as important virulence determinants for many bacteria and represent a promising carbohydrate source for glycoconjugate vaccines. In the Wzy-dependent O-antigen biosynthetic pathway, the integral membrane protein Wzy was shown to be the sole enzyme responsible for polymerization of O-repeat unit. Its catalytic mechanism, however, remains elusive. Herein, Wzy was successfully overexpressed in Escherichia coli with an N-terminal His₁₀-tag. Blue native polyacrylamide gel electrophoresis (BN-PAGE) revealed that the Wzy protein exists in its native confirmation as a dimer. Subsequently, we chemo-enzymatically synthesized the substrates of Wzy, the lipid-PP-linked repeat units. Together with an optimized O-antigen visualization method, we monitored the production of reaction intermediates at varying times. We present here our result as the first biochemical evidence that Wzy functions in a distributive manner.     

Gossypol toxicity and detoxification in Helicoverpa armigera and Heliothis virescens

Gossypol is a polyphenolic secondary metabolite produced by cotton plants, which is toxic to many organisms. Gossypol's aldehyde groups are especially reactive, forming Schiff bases with amino acids of proteins and cross-linking them, inhibiting enzyme activities and contributing to toxicity. Very little is known about gossypol's mode of action and its detoxification in cotton-feeding insects that can tolerate certain concentrations of this compound. Here, we tested the toxicity of gossypol and a gossypol derivative lacking free aldehyde groups (SB-gossypol) toward Helicoverpa armigera and Heliothis virescens, two important pests on cotton plants. Larval feeding studies with these two species on artificial diet supplemented with gossypol or SB-gossypol revealed no detectable toxicity of gossypol, when the aldehyde groups were absent. A cytochrome P450 enzyme, CYP6AE14, is upregulated in H. armigera feeding on gossypol, and has been claimed to directly detoxify gossypol. However, using in vitro assays with heterologously expressed CYP6AE14, no metabolites of gossypol were detected, and further studies suggest that gossypol is not a direct substrate of CYP6AE14. Furthermore, larvae feeding on many other plant toxins also upregulate CYP6AE14. Our data demonstrate that the aldehyde groups are critical for the toxicity of gossypol when ingested by H. armigera and H. virescens larvae, and suggest that CYP6AE14 is not directly involved in gossypol metabolism, but may play a role in the general stress response of H. armigera larvae toward plant toxins.     

Testing for viral contaminants of veterinary vaccines in Hungary

The safety of veterinary vaccines is of paramount importance and it is significantly jeopardised by extraneous agents such as bacteria, mycoplasma, Chlamydia and viruses. Several critical steps of vaccine manufacture involve a potential risk of viral contamination. Viruses, as extraneous, agents can be divided into two main groups. Group 1 agents, such as Pestivirus, chicken anaemia virus (CAV), and egg drop syndrome virus (EDSV) are well-known to manufacturers and authorities. Compendial detection methods, clear guidelines and legislation have been established to minimise the risk of contamination with these agents. Contrary to group 1, group 2 agents like Torque Teno virus (TTV) or RD114, a replication-competent feline γ-retrovirus, have only recently been recognised and their role as contaminants needs further investigation.Randomly selected veterinary vaccines used between 1992 and 2009 were tested by nucleic acid amplification for CAV, EDSV, and TTV. Pestivirus contamination was examined in 33 vaccines used between 1996 and 2006 and a further 27 vaccines used between 2007 and 2009 based on random selection of these vaccines. In addition to random tests done on vaccines used from 2007 on, 12 batches of live Aujeszky's disease vaccines submitted to our laboratory for Official Control Authority Batch Release (OCABR) were also tested for Pestivirus.     

肺炎链球菌糖疫苗的研究进展

肺炎链球菌(Streptococcus pneumoniae)是引起多种疾病的主要病原体,包括侵袭性感染(如败血症和脑膜炎菌血症),以及更常见的粘膜部位感染(如肺炎、中耳炎和鼻窦炎).根据肺炎链球菌表面荚膜多糖结构的不同可以分成不同的血清型,至今已经鉴定出98种,其中有20种具有高毒力.为了预防肺炎链球菌感染,已研制出...     

The combined recombinant cathepsin L1H and cathepsin B3 vaccine against Fasciola gigantica infection

Protections against Fasciola gigantica infection in mice immunized with the individual and combined cathepsin L1H and cathepsin B3 vaccines were assessed. The vaccines comprised recombinant (r) pro-proteins of cathepsin L1H and B3 (rproFgCatL1H and rproFgCatB3) and combined proteins which were expressed in Pichia pastoris. The experimental trials were performed in ICR mice (n = 10 per group) by subcutaneous injection with 50 μg of the recombinant proteins combined with Alum or Freund's adjuvants. At two weeks after the third immunization, mice were infected with 15 F. gigantica metacercariae per mouse by oral route. The percents of protection of rproFgCatL1H, rproFgCatB3 and combined vaccines against F. gigantica were approximately 58.8 to 75.0% when compared with adjuvant-infected control. These protective effects were similar among groups receiving vaccines with Alum or Freund's adjuvants. By determining the levels of IgG1 and IgG2a in the immune sera, which are indicative of Th1 and Th2 immune responses, it was found that both Th1 and Th2 humoral immune responses were significantly increased in vaccinated groups compared with the control groups, with higher levels of IgG1 (Th2) than IgG2a (Th1). Mice in vaccinated groups showed reduction in liver pathological lesions when compared with control groups. This study indicates that the combined rproFgCatB3 and rproFgCatL1H vaccine had a high protective potential than a single a vaccine, with Alum and Freund's adjuvants showing similar level of protection. These results can serve as guidelines for the testing of this F. gigantica vaccine in larger economic animals.     

Preparation and characterization of chitosans carrying aldehyde functions generated by nitrogen oxides

In this work, aldehyde-functionalized chitosan is produced by the reaction of chitosan with nitrogen oxides generated in situ from a HNO₃/H₃PO₄-NaNO₂ mixture. This method is more advantageous than the existing approaches, since the depolymerization is slower and the purification process is straightforward. The appearance of characteristic peaks in the Fourier transform infrared and carbon-13 nuclear magnetic resonance spectra (1733 cm⁻¹ and 183.4 ppm, respectively) of the product confirms the presence of the aldehyde functionality in the modified chitosans. The ¹H NMR spectra also revealed the presence of aldehyde groups. Furthermore, the gradual disappearance of the peaks due to aldehyde protons and a concomitant appearance of a new resonance at ∼8.05 ppm with increasing pH indicate the formation of Schiff's base between the aldehyde and the free amine groups. The aldehyde-functionalized chitosan prepared with 6 h of reaction time (chitosan-6h) forms a gel in situ without any added external crosslinker and it may potentially be useful as a vehicle for drug delivery.     

Comparison of O-Antigen Gene Clusters of All O-Serogroups of Escherichia coli and Proposal for Adopting a New Nomenclature for O-Typing

Escherichia coli strains are classified based on O-antigens that are components of the lipopolysaccharide (LPS) in the cell envelope. O-antigens are important virulence factors, targets of both the innate and adaptive immune system, and play a role in host-pathogen interactions. Because they are highly immunogenic and display antigenic specificity unique for each strain, O-antigens are the biomarkers for designating O-types. Immunologically, 185 O-serogroups and 11 OX-groups exist for classification. Conventional serotyping for O-typing entails agglutination reactions between the O-antigen and antisera generated against each O-group. The procedure is labor intensive, not always accurate, and exhibits equivocal results. In this report, we present the sequences of 71 O-antigen gene clusters (O-AGC) and a comparison of all 196 O- and OX-groups. Many of the designated O-types, applied for classification over several decades, exhibited similar nucleotide sequences of the O-AGCs and cross-reacted serologically. Some O-AGCs carried insertion sequences and others had only a few nucleotide differences between them. Thus, based on these findings, it is proposed that several of the E. coli O-groups may be merged. Knowledge of the O-AGC sequences facilitates the development of molecular diagnostic platforms that are rapid, accurate, and reliable that can replace conventional serotyping. Additionally, with the scientific knowledge presented, new frontiers in the discovery of biomarkers, understanding the roles of O-antigens in the innate and adaptive immune system and pathogenesis, the development of glycoconjugate vaccines, and other investigations, can be explored.     

Characterization of Ectromelia Virus Deficient in EVM036, the Homolog of Vaccinia virus F13L,and Its Application for Rapid Generation of Recombinant Viruses

The orthopoxvirus (OPV) vaccinia virus (VACV) requires an intact F13L gene to produce enveloped virions (EV) and to form plaques in cell monolayers. Simultaneous introduction of an exogenous gene and F13L into F13L-deficient VACV results in expression of the foreign gene and restoration of plaque size. This is used as a method to rapidly generate VACV recombinants without the need for drug selection. However, whether other OPVs require the orthologs of F13L to generate EV and form plaques, whether F13L orthologs and EV are important for OPV pathogenesis in natural hosts, and whether a system based on F13L ortholog deficiency can be used to generate recombinant OPVs other than VACV have not been reported. The F13L ortholog in ectromelia virus (ECTV), the agent of mousepox, is EVM036. We show that ECTV lacking EVM036 formed small plaques and was highly attenuated in vivo but still induced strong antibody responses. Reintroduction of EVM036 in tandem with the DsRed gene resulted in a virus that expressed DsRed in infected cells but was indistinguishable from wild-type ECTV in terms of plaque size and in vivo virulence. Thus, our data show that, like F13L in VACV, EVM036 is required for ECTV plaque formation and that EVM036 and EV are important for ECTV virulence. Our experiments also suggest that OPVs deficient in F13L orthologs could serve as safer anti-OPV vaccines. Further, our results demonstrate that ECTV deficient in EVM036 can be exploited for the rapid generation of fully virulent ECTV expressing foreign genes of interest.