Immunogenicity and Protective Efficacy of a Polyvalent DNA Vaccine against Human Orthopoxvirus Infections Based on Smallpox Virus Genes

DNA vaccines combining plasmids carrying the variola virus genes M1R, A30L, and F8L of intracellular virion surface membrane proteins as well as A36R and B7R of the extracellular virus envelope proteins under control of Rous sarcoma virus or cytomegalovirus promoters have been constructed.ese DNA vaccines induced production of a high titers of vaccinia virus-neutralizing antibodies in mice similar to those elicited by the live vaccinia virus immunization. Mice vaccinated by created DNA vaccine were completely protected against a lethal (10 LD50) challenge with highly pathogenic ectromelia virus. ese results suggest that such vaccine should be efficient in immunization of humans against smallpox.


Introduction
e historically �rst method used for human protection against devastating smallpox epidemics was the so-called variolation, that is, an intracutaneous inoculation of healthy persons with infectious material from human smallpox cases.e disease thus induced had a shorter incubation period and a milder course as compared with the disease caused by a common respiratory transmission of this infection.e mortality rate aer variolation was 0.5−2% versus 20−30% typical for the smallpox epidemics.e discovery of human vaccination using �rst cowpox virus (CPXV) and then vaccinia virus (VACV) had led to a considerable decrease in the severe side reactions [1,2].
Taking into account the postvaccination complications caused by classical live vaccine involving VACV and con-�rmation of the global smallpox eradication, the �orld Health Organization recommended in 1980 to stop further vaccination against this infection [1].Subsequent worldwide cessation of the vaccination against smallpox has created a most dangerous situation when the human population year by year becomes ever more unprotected not only from a potential infection with variola virus (VARV) as a result of a bioterrorism attack or reemergence of the virus in nature, but also from infection with other closely related orthopoxviruses, the natural reservoir of which is small rodents [3][4][5].is is demonstrated by more frequent outbreaks of human orthopoxvirus infections caused by monkeypox virus (MPXV), CPXV, and VACV [6][7][8][9][10].e absence of efficient antivirals makes the vaccine prevention the most important speci�c tool for control of the orthopoxvirus infections among humans.
Use of the classical live VACV vaccine for mass vaccinations is now unacceptable because of a relatively large number of potential complications, especially taking into account the increased number of population cohorts with suppressed immunity, such as the patients aer transplantation, HIV infected persons, and those taking immunodepressants.Correspondingly, it is especially topical challenge to design stateof-the-art safe vaccines against smallpox and other human orthopoxvirus infections.A promising direction is creation of a DNA vaccine, the newest approach to immune prevention of virus infections [11][12][13].Such vaccines are able to induce a full-�edged immune response to a viral infection being completely safe for humans.
Although the viruses belonging to the genus Orthopoxvirus are closely related and the vaccination of animals or humans with a member of this genus provides protection against infection with other orthopoxvirus species, it is known that the persons that had smallpox acquire a lifelong immunity, whereas the vaccination with vaccinia virus requires repeated immunizations with a certain periodicity to ensure no subsequent decline of immunity against smallpox [1,3].
e accumulated data on the main orthopoxvirus protective virion proteins and genome sequences have formed the background for designing a polyvalent DNA vaccine with a combination of virus genes [3,14].A considerable success in development of the DNA vaccines against orthopoxvirus infections has been demonstrated in several works where the mixtures of two-to-�ve plasmids carrying individual VACV genes have been used for vaccination and the protective effect has been assessed in the mice challenged with a lethal VACV dose [15][16][17].A study of a combined DNA vaccine involving four VACV genes using a macaque rhesus model challenged with MPXV aer vaccination has demonstrated an incomplete protective effect [18].is suggested that use of the genes of the corresponding orthopoxvirus in DNA vaccines could provide a better protection from the orthopoxvirus infection.Consequently, the DNA vaccine variants and subunit protein vaccine based on MPXV genes have been designed and tested in a macaque rhesus model [19].In this case, a protective effect from the lethal infection of the animals with MPXV was detected aer joint administration of both the DNA and protein vaccines.
Other authors have recently synthesized codon-optimized variants of three VARV genes for an anti-smallpox vaccine [20]; however, immunization of mice with such vaccine failed to provide a complete protection from a lethal challenge with VACV.
e goal of this work was to elaborate a combined DNA vaccine based on �ve natural VARV genes and to study its protective efficacy in the model of mice infected with ectromelia virus (ECTV), highly virulent for these animals.

Materials and Methods
2.1.Viruses and Cells.E. coli strain XL2-Blue, culture of African green monkey kidney epithelial cells Vero, vaccinia virus strain LIVP (VACV-LIVP), and ectromelia virus K1 strain (ECTV-K1) were obtained from the collection of the State Research Center of Virology and Biotechnology Vector.

Administration of DNA Vaccines.
Six-week-old female BALB/c mice were each immunized (intracutaneously, subcutaneously, intraperitoneally, or intramuscularly) with 100 g of endotoxin-free plasmid DNA in 100 L of PBS (in the case of a plasmid mixture, 50 g of each plasmid with a total dose of 250 g were used).e vaccines were administered three times with 3-week intervals; blood samples were taken 2 weeks aer immunization.Animal experimentation guidelines were followed in animal studies.

Plaque Reduction Neutralization
Assay.e sera from immunized animals (1/100 dilutions) were incubated with 100 PFU (plaque forming units) of VACV-LIVP for 1 h at 37 ∘ C. Con�uent Vero cell monolayers were infected with antibody-virus mixtures for 1 h, washed with PBS, and incubated under liquid overlay for 3 days.Aer removing the liquid, the monolayers were stained with 0.2% crystal violet for 10 min to count the plaques.e neutralization was calculated as the percent of the plaque counts, reduced in the tests with serum, relative to the number of the plaques in the virus control (without sera).

Challenge Experiment.
Aer three vaccine doses were administered at intervals of 3 weeks, mice were intraperitoneally challenged with 10 LD 50 (3.2× 10 2 PFU) of ECTV-K1.e challenge took place 3 weeks aer the last immunization.e mice were observed daily during 2 weeks.Animal experimentation guidelines were followed in animal studies.

VARV gene
Primer sequence Amplicon size (bp) DNA vaccine computed using the ADEPT version 2.1 program [34].Subsequent analysis has demonstrated that some VARV proteins insigni�cantly differ in their potential antigenic determinants from their VACV orthologs, namely, A30L and M1R, in one potential antigenic determinant and F8L, in two determinants, whereas the products of VARV genes A36R and B7R display considerable distinctions in the number of their potential antigenic determinants from their orthologs of other orthopoxvirus species (Table 2).Distribution of such discrepancies in amino acid sequence of A36R protein is given as an example in Figure 1.

e Optimal Method for DNA Vaccine Administration Depends on the Used Promoter for Eukaryotic Expression.
e intraperitoneal immunization of BALB/c mice with the DNA vaccines based on individual VARV genes A4L, A30L, A36R, B7R, F8L, I5R, M1R, and M4R demonstrated that the plasmid pBKRSV-F8L had the most pronounced immunogenic effect [35].It is known that the administration route and used eukaryotic promoter can in�uence the DNA vaccine efficacy [36].erefore, we used the plasmids pBKRSV-F8L and pcDNA-F8L for immunizing mice at a dose of 100 g/mouse using different administration routes, namely, intraperitoneal, intramuscular, intracutaneous, or subcutaneous.In two groups for negative and positive control, the mice were intraperitoneally immunized with physiological saline solution and VACV at a dose of 10 6 PFU per mouse, respectively.All preparations were applied three times with 3-week intervals to intraperitoneally challenge of 10-11 mice in each group with ECTV-K1 at a dose of 10 LD 50 3 weeks aer the last immunization.e best results were obtained for intracutaneous immunization with the DNA vaccines pcDNA-F8L (40% of survivals; Figure 2(a)) and intraperitoneal administration of the DNA vaccine pBKRSV-F8L (45% of survivals; Figure 2(b)).e combinations pcDNA-F8L/intraperitoneally, pcDNA-F8L/subcutaneously, pBKRSV-F8L/intracutaneously, or pBKRSV-F8L/subcutaneously gave considerably lower protection levels, namely, 9, 18, 18, and 9%, respectively.No survived animals were recorded in the groups intramuscularly immunized with the DNA vaccines pBKRSV-F8L or pcDNA-F8L.

Immunization with Individual IMV Genes Provides a Partial
Protection.Several groups were intracutaneously immunized at a dose of 100 g/mouse with the single-gene DNA vaccines pcDNA-A30L, pcDNA-F8L, or pcDNA-M1R as well as the control plasmid pcDNA and intraperitoneally with VACV at a dose of 10 6 PFU/mouse as negative and positive controls, respectively.e vaccines were administered three times with 3-week intervals to intraperitoneally challenge of 30-32 mice in each group with ECTV-K1 at a dose of 10 LD 50 .is experiment has demonstrated that the VARV (IMV) genes F8L, A30L, and M1R within the vector pcDNA administered intracutaneously provide 40, 33, and 25% protection, respectively, from the challenge with 10 LD 50 of ECTV (Figure 3).e observed protective efficacies of the DNA vaccines involving individual VARV (IMV) genes correlated with the virus-neutralizing activities of the sera: VARV genes F8L, A30L, and M1R within the vector pcDNA administered intracutaneously induced 57.0 ± 3.6, 52.0 ± 4.0 and 40.0 ± 2.5% level of neutralizing antibodies against VACV, respectively.

e Polyvalent DNA Vaccine Involving the VARV Genes A30L, M1R, F8L, A36R, and B7R Induces Production of High Titers of VARV-Neutralizing Antibodies and Provides
a Complete Protection against the Challenge with a Lethal ECTV Dose.Based on the results of previous studies [15,17,18,37], the following immunization schemes for the animal groups were used to assess the efficacy of polyvalent DNA vaccine: (1) intracutaneous immunization with a mixture of the �ve plasmids pcDNA-A30L, pcDNA-F8L, pcDNA-M1R, pcDNA-A36R, and pcDNA-B7R, 50 g each (a total dose of 250 g/mouse); (2) intraperitoneal immunization with a mixture of the plasmids pBKRSV-A30L, pBKRSV-F8L, pBKRSV-M1R, pBKRSV-A36R, and pBKRSV-B7R, 50 g each (a total dose of 250 g/mouse); (3) intracutaneous immunization with the plasmid pcDNA at a dose of 250 g/mouse (negative antibodies (Figure 4).Note that the level of neutralizing antibodies aer the third immunization with DNA vaccines was comparable with the level of neutralizing antibodies aer immunization with the live VACV, 90.0 ± 5.0% (the data are shown for the mouse serum titer of 1/100).e polyvalent DNA vaccine based on the vector pcDNA induced a similar level of neutralizing antibodies (87.0 ± 4.0%) as compared with the vaccine based on the vector pBKRSV (80.0 ± 2.5%).
When assessing the protective efficacy, the immunized mice were challenged with ECTV-K1 at a dose of 10 LD 50 with subsequent observation for 14 days (Figure 5).In negative control groups, 90% of the mice immunized with pBKRSV and 100% of the animals immunized with pcDNA died by the end of the observation period.No mice died by the end of the observation period in the group immunized with live VACV.Both variants of the polyvalent DNA vaccine involving the VARV genes A30L, F8L, M1R, A36R, and B7R also provided a complete protection of the mice from the challenge with a lethal ECTV dose.

Discussion
It is known that only those persons who had smallpox acquire almost a lifelong immunity to this disease, whereas those who were vaccinated with VACV require repeated immunizations with a certain periodicity to maintain their antismallpox immune status [1,3].A reliable protection against orthopoxvirus infection implies a concurrent induction of the immune responses to two infective forms of the virus, intracellular and extracellular virions, which is achievable only when using the immunogens of intracellular virion surface membrane and extracellular virus envelope within one vaccine [15,17,18,37].
A high potential of the VARV genes A30L, M1R, and F8L, encoding intracellular virion surface membrane proteins, and the genes A36R and B7R, encoding the extracellular virus envelope proteins, in development of the anti-smallpox DNA vaccines has been recently shown for their VACV orthologs in protection of mice against VACV infection [16,37].At the �rst stage of the work, we compared the structures of these immunodominant proteins of the orthopoxviruses pathogenic for humans (VARV, MPXV, CPXV, VACV) with the same proteins of ECTV, nonpathogenic for humans but highly pathogenic for mice.We have used a previously designed ADEPT soware program which localizes potential B cell epitopes in analyzed proteins [34].It uses a matrix of amino acid values to calculate moving average along the sequence.e matrix contains a value for each amino acid which shows how much an amino acid contributes to the region being potential B cell epitope.e matrix was created by running the program in learning mode on the database of well-known proteins.In the case of orthopoxviral proteins, these potential epitopes have not been con�rmed experimentally but were analyzed using ADEPT program for understanding that the DNA vaccine involving the VARV genes should be more efficacious as compared with the vaccine based on VACV genes in inducing the protective immunity of humans to smallpox.Despite that orthopoxviruses differ in the number (Table 2) and location (Figure 1) of their potential antigenic determinants, immunization of animals with a representative of one of the species induces a cross-immune response provided for protection against other orthopoxvirus species [1].Although this protection is suboptimal, it is still possible to conduct preclinical trials of the developed vaccines against smallpox using the models of small laboratory animals and the orthopoxviruses pathogenic for them.e most highly pathogenic virus for mice is ECTV, which we correspondingly selected for assessing a protective efficacy of the developed DNA vaccine.
It is known that the efficacy of a DNA vaccine depends to a considerable degree on various parameters, such as the used antigen, structure of vector plasmid, promoter for eukaryotic expression of the target antigen, administration route, amount of plasmid, and number of immunizations, since all these factors in�uence the strength and quality of the induced immune response [36].Correspondingly, aiming to increase the efficacy of the elaborated DNA vaccine, we have analyzed the combination of administration route and used eukaryotic promoter by the example of VARV F8L gene to select the optimal variant providing for induction of the most pronounced protective immune response to orthopoxvirus infection.is has allowed us to discover that the DNA vaccine carrying the promoter from RSV (pBKRSV) is the most efficient in the case of intraperitoneal administration (45% of survivals), while the DNA vaccine with the CMV promoter (pcDNA) and the same gene of VARV immunodominant protein is the most efficient in the case of intracutaneous administration (40% of survivals).Other combinations of rate immunization and type of DNA vaccine gave no more than 18% of survivals.is allows us to give a conclusion about the reliability of obtained results.It has been earlier repeatedly demonstrated that the efficiencies of these two promoters in inducing the immune response to the protein product of a target gene depends on particular experimental conditions.For example, it has been shown in the work on designing the DNA vaccine against in�uenza virus based on the virus nucleoprotein gene that the CMV promoter provides a stronger humoral response in the case of an intracutaneous administration as compared with the RSV promoter, although both promoters induce equal immune responses when the DNA vaccine is administered intramuscularly [38].
At the next stage of our work, we have demonstrated that the DNA vaccines based on the individual genes M1R, A30L, or F8L, encoding VARV intracellular virion surface membrane proteins, under the control of CMV promoter being intracutaneously administered induce production of VACV-neutralizing antibodies and provide for a partial mouse protection against ECTV challenge at a dose of 10 LD 50 (Figure 3).Earlier Hooper et al. [16] have demonstrated that the DNA vaccines based on two EEV proteins, encoded by A33R and B5R VACV (orthologs to A36R and B7R VARV), provide for 80 % mouse protection against VACV WR challenge at a dose of 12.5 LD 50 .At the same time combination of two IMV proteins, encoded by A27L and L1R, and two EEV proteins, encoded by A33R and B5R VACV (orthologs to A30L, M1R, A36R, and B7R VARV), provide for 100% mouse protection against VACV WR challenge at a dose of 12.5 LD 50 [16].
Using a combined variant of the DNA vaccine involving �ve plasmids carrying the genes M1R, A30L, and F8L of intracellular virion surface membrane proteins as well as A36R and B7R of the extracellular virus envelope proteins, it has been shown that both variants of the DNA vaccine-the variant with the vector pBKRSV administered intraperitoneally and the variant with the vector pcDNA administered intracutaneously-induce production of a high titers of VACV-neutralizing antibodies similar to the live VACV vaccine (Figure 4) and provide a complete protection of the animals against a lethal dose of highly pathogenic ECTV (Figure 5).e considerable differences of the used VARV antigens from their ECTV orthologs in both the number and localization of their potential antigenic determinants (Table 2) as well as the observed protective effect to lethal ECTV challenge aer immunization with the elaborated combined DNA vaccines suggest that such vaccine should be efficient in immunization of humans against smallpox.Using of the developed DNA vaccine for prime-immunization of humans against smallpox with following boost-immunization with VACV will allow avoid a side effects characteristic to classic alive smallpox vaccine used alone [2].

T 1 :
Primers used to amplify VARV genes by PCR.

F 2 :F 3 :F 4 :
e survival rates of BALB/c mice aer triple intraperitoneal (i/p), intramuscular (i/m), intracutaneous (i/c), or subcutaneous (s/c) immunization with (a) pcDNA-F8L or (b) pBKRSV-F8L and intraperitoneal challenge with ectromelia virus strain K1 at a dose of 10 LD 50 .10-11 mice in each group were challenged 3 weeks aer the last immunization.e survival rates of BALB/c mice aer triple intracutaneous immunization with the DNA vaccines pcDNA-A30L, pcDNA-F8L, or pcDNA-M1R and intraperitoneal challenge with ectromelia virus strain K1 at a dose of 10 LD 50 .30-32 mice in each group were challenged 3 weeks aer the last immunization.control no.1); (4) intraperitoneal immunization with the plasmid pBKRSV at a dose of 250 g/mouse (negative control no. 2) and (5) intraperitoneal immunization with VACV at a dose of 10 6 PFU/mouse (positive control).e vaccines were administered with 3-week intervals; 3 weeks aer the last immunization, 30 mice in each group were intraperitoneally challenged with ECTV-K1 at a dose of 10 LD 50 .eimmunizations with both variants of the polyvalent DNA vaccine induced production of the VACV-neutralizing Dynamics of the level of virus-neutralizing antibodies during intracutaneous immunization of BALB/c mice with the polyvalent DNA vaccine pcDNA-A30L, pcDNA-F8L, pcDNA-M1R, pcDNA-A36R, and pcDNA-B7R (pcDNA-Σ5); intraperitoneally with the polyvalent DNA vaccine pBKRSV-A30L, pBKRSV-F8L, pBKRSV-M1R, pBKRSV-A36R, and pBKRSV-B7R (pBKRSV-Σ5); or intraperitoneally with VACV detected by VACV neutralization in Vero cell culture.Mean values of the neutralization (%) for serum dilution of 1 : 100 ± standard deviations are shown.Numbers 1, 2, and 3 indicates the number of immunizations.
AsuNHI (GCTAGC) and SalI (GTCGAC) sites for cloning sequence are boldfaced; initiation and stop codons are underlined; a Kozak sequence added between the initiation codon and protein sequence is italicized.
Comparison of the amino acid sequence of the VARV A36R protein with the corresponding VACV, CPXV, MPXV, and ECTV orthologs.Identical amino acids in the compared sequence of virus genomes relative to the VARV strain India-1967 are denoted with dots and deletions, with dashes.e amino acid positions in analyzed segment are shown above the amino acid sequence.