Current address: Department of Pediatrics, Kansai Rousai Hospital, Hyogo, Japan.
Current address: Astellas Pharma Inc., Tokyo, Japan.
Current address: Department of Biology, High Point University, High Point, North Carolina, United States of America.
Current address: Harrisvaccines, Inc. Ames, Iowa, United States of America.
Conceived and designed the experiments: JIC. Performed the experiments: JS YH JJB TK PDB VMC KK. Analyzed the data: JS YH JJB PDB VMC KK JIC. Wrote the paper: JIC.
The authors have declared that no competing interests exist.
Epstein-Barr virus (EBV) is a human lymphocryptovirus that is associated with several malignancies. Elevated EBV DNA in the blood is observed in transplant recipients prior to, and at the time of post-transplant lymphoproliferative disease; thus, a vaccine that either prevents EBV infection or lowers the viral load might reduce certain EBV malignancies. Two major approaches have been suggested for an EBV vaccine- immunization with either EBV glycoprotein 350 (gp350) or EBV latency proteins (e.g. EBV nuclear antigens [EBNAs]). No comparative trials, however, have been performed. Rhesus lymphocryptovirus (LCV) encodes a homolog for each gene in EBV and infection of monkeys reproduces the clinical, immunologic, and virologic features of both acute and latent EBV infection. We vaccinated rhesus monkeys at 0, 4 and 12 weeks with (a) soluble rhesus LCV gp350, (b) virus-like replicon particles (VRPs) expressing rhesus LCV gp350, (c) VRPs expressing rhesus LCV gp350, EBNA-3A, and EBNA-3B, or (d) PBS. Animals vaccinated with soluble gp350 produced higher levels of antibody to the glycoprotein than those vaccinated with VRPs expressing gp350. Animals vaccinated with VRPs expressing EBNA-3A and EBNA-3B developed LCV-specific CD4 and CD8 T cell immunity to these proteins, while VRPs expressing gp350 did not induce detectable T cell immunity to gp350. After challenge with rhesus LCV, animals vaccinated with soluble rhesus LCV gp350 had the best level of protection against infection based on seroconversion, viral DNA, and viral RNA in the blood after challenge. Surprisingly, animals vaccinated with gp350 that became infected had the lowest LCV DNA loads in the blood at 23 months after challenge. These studies indicate that gp350 is critical for both protection against infection with rhesus LCV and for reducing the viral load in animals that become infected after challenge. Our results suggest that additional trials with soluble EBV gp350 alone, or in combination with other EBV proteins, should be considered to reduce EBV infection or virus-associated malignancies in humans.
Epstein-Barr virus (EBV) is the primary cause of infectious mononucleosis and is associated with several cancers. Presently there is no licensed vaccine to prevent EBV diseases. Two types of candidate vaccines are under development; one involves immunization with the major glycoprotein (gp350) on the outside of the virus, while the other involves vaccination with EBV proteins expressed during latency. We compared these two types of candidate vaccines in a rhesus monkey model of EBV and found that the gp350 vaccine induced better protection from infection. In addition, animals that received the rhesus EBV glycoprotein and became infected had a lower level of rhesus EBV DNA in the blood at 23 months after challenge than animals that received the rhesus EBV latency protein vaccine that subsequently were infected. Since levels of EBV DNA in the blood have been predictive for EBV lymphomas in transplant patients, the ability of rhesus EBV gp350 to reduce levels of rhesus EBV in the blood after infection suggests the EBV gp350 could have a role in reducing certain EBV-associated cancers. This is the first test of candidate vaccines in the rhesus monkey model of EBV and shows that this model should be useful in further evaluation of EBV vaccines.
Epstein-Barr virus (EBV) is a causative agent of infectious mononucleosis and is associated with a number of malignancies including lymphomas in immunocompromised persons, Hodgkin lymphoma, Burkitt lymphoma, and nasopharyngeal carcinoma. Currently no vaccine has been licensed to prevent EBV infection or disease.
Most attempts to generate an EBV vaccine have focused on glycoprotein 350 (gp350) as the immunogen. gp350 is the most abundant EBV glycoprotein in virions and on the surface of infected cells. gp350 binds to CD21, the EBV receptor on B cells. EBV gp350 is spliced to form gp220. gp350 is important for virus absorption to B cells and soluble gp350 can block EBV infection. Antibodies to gp350 neutralize virus in vitro
While gp350 is important for protection from infectious mononucleosis, EBV proteins expressed during latency are thought to be critical for controlling latent infection. The EBV nuclear antigen 3 (EBNA-3) latency proteins are the primary targets of CD8 T cells in the blood of healthy EBV carriers
Given the complexities and costs of EBV vaccine trials in humans, testing vaccines in animal models might allow more rapid comparison of candidate vaccines. Many animal studies using gp350 have been performed in cottontop tamarins, which have several limitations. These animals cannot be infected with EBV by the oral route, they do not develop a persistent infection similar to humans, and the animals do not express MHC class I A, B or C alleles
While EBV gp350 has been shown to be protective against tumors in cottontop tamarins challenged with high titers of EBV and one study showed that gp350 reduced the incidence of infectious mononucleosis in humans, no vaccine studies have been performed using rhesus LCV in monkeys. Furthermore no studies have been reported involving a direct comparison of different EBV vaccines, including gp350 versus EBV latency proteins, in the same trial.
We compared three rhesus LCV vaccines- (a) recombinant soluble rhesus LCV gp350, (b) rhesus LCV gp350 expressed from replication-defective, single cycle, virus-like replicon particles (VRPs) derived from an attenuated strain of Venezuelan equine encephalitis (VEE), and (c) a combination of rhesus LCV gp350, EBNA-3A, and EBNA-3B each expressed in separate attenuated VRPs for their ability to protect rhesus monkeys against infection with rhesus LCV and to determine their long term effect on rhesus LCV DNA in the blood after challenge.
These experiments were approved by the Animal Care and Use Committees of the National Institute of Allergy and Infectious Diseases and the University of California, Davis. The studies were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.
Rhesus macaques were reared separately from rhesus LCV seropositive animals beginning at birth and serologic testing indicated that all animals were seronegative for rhesus LCV. Six to 18 month old animals were housed in pairs during the vaccination period, and housed separately after challenge. Animals were vaccinated by inoculation in the triceps muscle, and challenged with rhesus LCV by inoculation of the back of the throat with virus in 1 ml of cell culture media using a needleless syringe.
Rhesus LCV was isolated from LCL8664 cells (American Type Culture Collection, Manassas, VA). The cells were derived from a rhesus monkey with a malignant lymphoma
Modified vaccinia Ankara (MVA) expressing rhesus LCV gp350 and green fluorescent protein (GFP) was constructed by cloning rhesus LCV gp350 into plasmid pLW44
The extracellular domain of rhesus LCV gp350 (amino acids 1–737
To construct pSGrhgp350, the rhesus LCV gp350 gene was amplified from LCL8664 cell DNA by PCR using primers rhgp350-F-EcoR containing an EcoR I site (
To measure antibody to rhesus LCV gp350, a plasmid was constructed containing the viral gene linked to the Renilla luciferase gene. The extracellular domain of the rhesus LCV gp350 gene was amplified by PCR using primers rhgp350-F-EcoR (see above) and rhgp350-TMR (
Since Jiang et al.
To clone EBNA-3A1 and EBNA-3A2 into an SV40 expression vector, PCR was performed with primer rhEBNA3A-FBcl
To clone EBNA-3A1 into a CMV expression vector, pLWrhEBNA3A was digested with Pst I, blunted with T4 DNA polymerase, digested with Sal I, and the fragment containing EBNA-3A was inserted into the Sal I-Sma I site of pCI to produce pCIrhEBNA3A.
Rhesus LCV EBNA-3B was amplified from LCL8664 cell DNA as two separate fragments using primer pairs rhEBNA3BF-Sal (
To clone EBNA-3B into a SV40 expressing vector, EBNA-3B was removed from plasmid pCRrhEBNA3B by digestion with Sal I and Pst I, the ends were blunted using T4 DNA polymerase and the EBNA-3B gene was inserted into the Bam HI site of plasmid pSG5 (after blunting with the Klenow fragment of DNA polymerase I) to obtain pSGrhEBNA3B. To clone EBNA-3B into a CMV expression vector, EBNA-3B was removed from plasmid pCRrhEBNA3B by digestion with Sal I and Not I and inserted into the corresponding sites of pCI to obtain pCIrhEBNA3B.
To remove the RBP-Jκ binding sites from rhEBNA-3A1 we deleted codons 204–207 (TFAC) corresponding to nucleotides 610–621 from pSGrhEBNA3A1. To remove the RBP-Jκ binding sites from rhEBNA-3B, we deleted codons 208 to 211 (TLGC) corresponding to nucleotides 622–633 from pSGrhEBNA3B using the Quik Change site-directed Mutagenesis kit (Stratagene). The resulting plasmids deleted for the EBNA-3 RBP-Jκ binding sites, pSGrhEBNA3A1-del, pSGrhEBNA3A2-del, and pSGrhEBNA3B-del, were only used for cloning into the Venezuelan equine encephalitis vector (see below). All gp350, EBNA-3A, and EBNA-3B constructs obtained by PCR were sequenced.
To produce rhesus LCV gp350-Fc protein, CV-1/EBNA-1 cells (ATCC, Manassas, VA) grown in DMEM/F-12 medium (1∶1) with 10% fetal bovine serum, were transfected with plasmid pDCrhgp350-Fc using DEAE-Dextran. After transfection, the media was changed to DMEM/F12 medium with 0.5% low immunoglobulin G fetal bovine sera (HyClone, Logan, UT). One week after transfection, the media was collected, clarified by low speed centrifugation, and filtered through a 0.45 um filter. Recombinant rhesus LCV gp350-Fc was bound to protein A-Sepharose beads, eluted from the beads with 12.5 mM citric acid pH 2.2, and collected in tubes containing 500 mM HEPES, pH 9.0 to neutralize the citric acid.
To produce recombinant rhesus LCV EBNA-3A and EBNA-3B, Cos cells were transfected with plasmid pSGrhEBNA3A1 or pSGrhEBNA3B using Lipofectamine 2000 (Invitrogen). Two days after transfection, lysates were prepared from the cells and proteins were separated by polyacrylamide gel electrophoresis. Proteins were stained with Coomassie blue, and bands containing EBNA-3A and EBNA-3B were excised from the gel. EBNA-3 proteins were eluted from the gel overnight in PBS and concentrated using a Centricon YM-100 filter (Millipore).
To produce antibody to rhesus LCV gp350, 2 rabbits were immunized with 150 ug of rhesus LCV gp350-Fc fusion protein in complete Freund's adjuvant (Animal Pharm Service Inc., Sausalito, CA). Animals were boosted with 100 ug of gp350-Fc in incomplete Freund's adjuvant on days 28, 42, and 86 after the first vaccination; 2 weeks after the last boost the rabbits were bled and sera were obtained.
To produce antibody to rhesus LCV EBNA-3A and EBNA-3B, mice were immunized three times, 3 weeks apart with 100 ug of pCIrhEBNA3A or pCIrhEBNA3B. Three weeks after the 3rd DNA immunization, the animals were boosted with 20 ug of EBNA-3A protein or 15 ug of EBNA-3B protein in complete Freund's adjuvant. Serum was collected from the mice 2 weeks later.
For rhesus monkey vaccinations, rhesus LCV gp350-Fc protein was incubated with Alhydrogel 2% (Brenntag Biosector, Accurate Chemical and Scientific Corp) by mixing on a rotating wheel for 30 min at room temperature followed by addition of monophosphoryl lipid A (Avanti Polar Lipids, Inc., Alabaster, AL).
Replication-defective attenuated Venezuelan equine encephalitis viruses (VEE) expressing rhesus LCV gp350, EBNA-3A1, or EBNA-3B were constructed by PCR amplification of the genes from plasmids pSGrhgp350, pSGrhEBNA3A1-del, pSGrhEBNA3B-del and inserting the rhesus LCV genes into a VEE replicon vector. The replicon vector contains the VEE nsP1, nsP2, nsP3, and nsP4 genes and an internal ribosome entry site (IRES) followed by a cloning site into which the rhesus LCV genes were inserted
Antibody to rhesus LCV viral capsid antigen (VCA) was determined by immunofluorescence (VRL Laboratories, San Antonio, Texas).
Antibody to rhesus LCV gp350 was measured using the luciferase immunoprecipitation system (LIPS) assay
DNA was isolated from 1−5×106 PBMCs using either a QIAamp DNA Blood Mini Kit (Qiagen) or an Easy-DNA Kit (Invitrogen). Real time PCR for rhesus LCV DNA was performed with primers and probes that amplify rhesus LCV EBER1
Real time reverse-transcriptase PCR was performed for rhesus LCV EBER1. Total RNA was isolated from 5×106 PBMCs using Trizol (Invitrogen, Calsbad, CA), and reverse transcription and PCR was performed using primers and probes as described previously
Rhesus monkey cell lines were used to present EBNA-3A, EBNA-3B, and gp350 to rhesus PBMCs. Lymphoblastoid cell lines (LCLs), which express EBNA-3A and EBNA-3B, were constructed for each monkey by infecting PBMCs with rhesus LCV in the presence of cyclosporine A (500 ng/mL, Sigma-Aldrich) and culturing the cells in RPMI 1640 with GlutaMax (Invitrogen) with 10% FBS and antibiotics. Cryopreserved PBMCs were thawed and cultured in RPMI 1640 with GlutaMax with 10% FBS, IL-2 (5 U/mL, from the National Cancer Institute) and antibiotics in 12-well plates overnight. The following day, PBMCs were divided into 2 tubes (1−3×106 cells per tube) and were cocultured with 2×106 autologous LCLs for 5 hours in the presence of 10 µg/mL of brefeldin A (Sigma-Aldrich) and 10 U/mL of IL-2. The cells were then washed in PBS with 2% FBS and 2 mM EDTA, incubated with FITC-conjugated anti-CD8 monoclonal antibody (clone RPA-T8, BioLegend, San Diego, CA) and APC-conjugated anti-CD4 monoclonal antibody (clone OKT4, BioLegend) for 20 min, washed with PBS containing 2% FBS and 2 mM EDTA, incubated with Cytofix/Cytoperm buffer (BD Bioscience, Franklin Lakes, NJ) for 25 min, and washed with Perm wash buffer (BD Bioscience). Cells were then incubated with PE-conjugated anti-IL-2 monoclonal antibody (clone MQ1-17H12, BioLegend) and PE-Cy7-conjugated anti-IFN-γ monoclonal antibody (clone B27, BD Bioscience), washed with Permwash buffer, and resuspended in PBS with 2% FBS and 2 mM EDTA. As a negative control, PBMCs were cultured without LCLs, and mixed with LCLs after fixation of PBMCs with Cytofix/Cytoperm buffer. Data were acquired using a FACS Caliber (BD Bioscience) and analysis was performed using Flowjo software 8.8.4 (Tree Star Inc., Ashland, OR). The percent of rhesus LCV-specific cytokine T cell response was defined as the percent cytokine (IL-2 or IFN-γ, or both) positive CD4 or CD8 cells in LCL-stimulated PBMCs minus the percent cytokine positive CD4 or CD8 cells in unstimulated PBMCs. If unstimulated samples had a higher frequency of cytokine positive cells than stimulated samples, a value of 0% was assigned, instead of a negative value.
To measure gp350-specific CD8 and CD4 T cell responses, LCLs were infected with either wild-type MVA, or MVA expressing rhesus LCV gp350, at 3 TCID50 for 24 hours before coculture with PBMCs. PBMCs were thawed and cultured overnight as described above. The following day, PBMCs were divided into 3 tubes (0.8−2×106 cells per tube) and cocultured with LCLs infected with either wild-type or gp350 expressing MVA for 5 hours in the presence of brefeldin A. As a negative control, PBMCs were cultured without LCLs and mixed with LCLs (not infected with MVA) after fixation of PBMCs with Cytofix/Cytoperm buffer. Staining and flow cytometry were done as described above. The percent of cytokine producing CD4 or CD8 cells in unstimulated PBMCs mixed with LCLs (not infected with MVA) after fixation (negative control) was subtracted from the percent of CD4 or CD8 cells in PBMCs stimulated with gp350 MVA-infected LCLs or wild-type MVA-infected LCLs. The percent of rhesus LCV gp350-specific CD4 or CD8 T cell response was defined as the percent of cytokine producing CD4 or CD8 cells in PBMCs stimulated with gp350 MVA-infected LCLs minus the percent of cytokine producing CD4 or CD8 cells in PBMCs stimulated with wild-type MVA-infected LCLs.
In order to determine if rhesus LCV encodes gp350 similar to its human EBV homolog, rabbits were immunized with purified rhesus LCV gp350-Fc fusion protein and serum was obtained. The rabbit serum detected proteins from 200−270 kDa in supernatant from cells transfected with plasmid expressing soluble gp350-Fc, but not with plasmid expressing GFP (pGL3-GFP)
Supernatant from cells transfected with control plasmid pGL3-GFP, or lysates from cells infected with MVA or VRP-GFP are negative controls.
To ensure that the rabbit antibody was specific for rhesus LCV gp350, we determined that the antibody could detect full length gp350 in virus-infected cells. Full length rhesus LCV gp350 was inserted into modified vaccinia Ankara (MVA). DF-1 cells were infected with MVA-gp350GFP or MVA alone and 16−24 hr later, lysates were prepared, and immunoblotted with the rabbit serum. Cells infected with MVA-gp350GFP, but not MVA alone produced a 250 kDa protein that reacted with the antibody
In order to express rhesus LCV EBNA-3A and EBNA-3B, we cloned the genes from LCL8664 cells into expression vectors and determined the sequence of the viral genes. While the sequence of rhesus LCV EBNA-3A was identical to the published sequence
Numbers indicate amino acid positions of rhesus LCV EBNA-3B and arrows indicate where the sequences of rhesus LCV EBNA-3B diverge and then return to identity.
Both EBV EBNA-3A
Based on the sequence of rhesus LCV EBNA3A (rhEBNA3A), either of two methionines could be the first amino acid of the protein
(A) Cos cells transfected with pSGrhEBNA3A1 and pSGrhEBNA3A1-del express EBNA-3A beginning at the first methionine without (lane 2) or with (lane 3) a deletion in the putative RBP-Jκ binding site, respectively. Cos cells transfected with pSGrhEBNA3A2 and pSGrhEBNA3A2-del express EBNA-3A beginning at the second methionine without (lane 4) or with (lane 5) a deletion in the putative RBP-Jκ binding site, respectively. Cos cells were transfected with pSGrhEBNA3B and pSGrhEBNA3B-del, with a deletion in the putative RBP-Jκ binding site (lanes 6 and 7, respectively). Cos cells were transfected with empty vector (pSG5, lane 8). (B) Detection of EBNA-3A in Vero cells infected with VRP-EBNA-3A, or in LCL8664 cells and in a rhesus monkey LCL (LCL-V). Arrow indicates location of EBNA-3A. (C) Detection of EBNA-3B in Vero cells infected with VRP-EBNA-3B, or in LCL8664 cells and LCL-V. Arrow indicates location of EBNA-3B. Additional bands noted in cells infected with VRP expressing EBNA-3A or -3B are likely due to overexpression of the protein.
Four rhesus LCV seronegative monkeys each received one of four inocula intramuscularly: (a) 50 ug of rhesus LCV soluble gp350-Fc protein (soluble gp350) formulated in 800 ug alum and 50 ug monophosphoryl lipid A, (b) 1×108 infectious units (IU) of virus-like replication-defective VEE particles expressing rhesus LCV gp350 (VRP-gp350) in 1 ml of DMEM with 10% FBS, (c) a combination of three separate replication-defective VEE particles expressing rhesus LCV gp350 (VRP-gp350), EBNA-3A (VRP-EBNA-3A), and EBNA-3B (VRP-EBNA-3B) each at a titer of 1×108 IU in a total of 1 ml of DMEM with 10% FBS, or (d) PBS control. The rhesus LCV soluble gp350 used in our vaccine contains the extracellular domain of the glycoprotein fused to the Fc domain of human IgG, while the vaccine used in the large human trial
Serum antibody responses to gp350 in animals 5 weeks after the last vaccination showed that all animals vaccinated with soluble gp350 or VRP-gp350 (alone or in combination with VRPs expressing EBNA-3A and EBNA-3B) produced antibodies to the glycoprotein (
gp350 antibody levels in naturally infected monkeys are shown. Antibody levels are measured as luminometer units. Cut off value is shown as horizontal dotted line, which was determined as the mean +2 standard deviations of the blank signal (open circles). Horizontal bars indicate geometric mean, asterisks indicate p<0.05 (Mann-Whitney's U-test), NS indicates not significant.
Rhesus LCV LCL-specific CD4 and CD8 T cell immune responses in monkeys were measured both pre- and post-vaccination. Rhesus LCV LCLs, which express EBNA-3A and EBNA-3B (
Animals were immunized with soluble gp350, VRP-gp350, a combination of VRP-gp350, VRP-EBNA-3A, and VRP-EBNA-3B, or PBS before challenge with wild-type virus. Pre indicates PBMCs obtained before vaccination; post indicates PBMCs obtained after vaccination.
We did not observe CD4 or CD8 T cell responses to rhesus LCV gp350 in monkeys after vaccination using LCLs as antigen presenting cells, except for one animal that had an increase in rhesus LCV LCL-specific cytokine positive CD4 T cell response after vaccination with VRP-gp350 (
A challenge inoculum of rhesus LCV was titered in LCV seronegative rhesus monkeys. Five animals were initially given 14 TID50 (infectious dose of virus needed to transform 50% of wells of cells in vitro) of rhesus LCV by application of virus to the throat and all animals seroconverted. Based on these results, 10 weeks after the last vaccination, animals were challenged by the oral route with 50 TID50 of rhesus LCV.
Antibody to rhesus LCV viral capsid antigen (VCA) was detected after challenge with rhesus LCV in all 4 animals that received PBS and all 4 that received VRP-gp350. In contrast, 2 of 4 that received soluble gp350 and 3 of 4 animals that received a combination of VRP-gp350, VRP-EBNA-3A, and VRP-EBNA-3B developed antibody to rhesus LCV VCA after challenge (
Like EBV, rhesus LCV DNA is present at very low or undetectable copy numbers in PBMCs of healthy animals infected in the past, but is usually detected in the blood after initial infection
Real time PCR was performed using a probe that detects EBER1 DNA.
To further verify that animals were protected from infection after challenge we tested PBMCs from animals for rhesus LCV EBER1. EBER1 is present in thousands of copies in virus-infected B cells and is usually detected in the blood for life after infection of rhesus monkeys
RNA was isolated from PBMCs and reverse transcription was performed followed by real time PCR with a probe that detects EBER1 DNA.
In summary, animals receiving soluble gp350 had the best level of protection after challenge with the fewest numbers of animals with rhesus LCV DNA or rhesus LCV RNA in the blood and the lowest rate of seroconversion after challenge, while animals that received the combination of VRP-gp350, VRP-EBNA-3A, and VRP-EBNA-3B had the next best level of protection.
Analysis of fever, lymph node swelling, liver function tests, and CD4 to CD8 ratios after challenge did not show discernable differences in animals that received different vaccines or control PBS (data not shown). This may not be surprising since there were small numbers of animals, all animals were all <3 years old (and EBV infectious mononucleosis is rare in young children), and variability in animals after infection has been reported previously
PBMCs were obtained from each of the vaccinated animals at 23 and 34 months after challenge. Using real time PCR with the rhesus LCV DNA EBER1 probe, we were only able to detect rhesus LCV DNA in PBMCs from 1 of the 16 animals 23 months after challenge (data not shown). Therefore, we developed a more sensitive real time PCR assay using IR1 DNA (corresponding to the Bam HI W repeats of EBV) that are present at 5.7 copies in the rhesus LCV genome
Blood was obtained 23 (A) and 34 months (B) after challenge. Real time PCR was performed using a probe that detects rhesus LCV IR1 (corresponding to the EBV Bam HI W repeat) DNA. Horizontal lines indicate mean values.
Two different types of vaccines have been developed to prevent disease and limit primary infection with EBV. Soluble EBV gp350 reduced the rate of infectious mononucleosis by 78% in young adults
Animals vaccinated with soluble gp350 produced the highest levels of antibody to the glycoprotein and these levels were higher than those seen in monkeys naturally infected with rhesus LCV. Prior studies have shown that antibody to gp350 is likely the predominant component of neutralizing antibody to EBV
We compared soluble rhesus LCV gp350 with VRPs expressing gp350 with the expectation that expression of the viral glycoprotein in cells infected with VRPs might enhance the immunogenicity of gp350 beyond its ability to induce antibody. Animal studies have shown that neutralizing antibody to gp350 alone does not always correlate with protection from disease. When cottontop tamarins were vaccinated with replication-defective adenovirus expressing gp350, non-neutralizing antibody to gp350 was induced, but the animals were protected against lymphoma
Animals vaccinated with alphavirus VRPs expressing EBNA-3A and EBNA-3B developed CD4 and CD8 cell responses to these proteins, while those vaccinated with VRPs expressing gp350 did not have detectable cellular responses to the glycoprotein. It is possible that the different methods used to present these antigens (LCLs naturally expressing EBNA-3A and EBNA-3B versus cells infected with MVA expressing gp350) could be responsible for these differences. Alphavirus VRPs target dendritic cells, which are highly efficient antigen presenting cells, and are effective for inducing cellular immunity
After challenge of animals with rhesus LCV, animals vaccinated with soluble rhesus LCV gp350 had the best level of protection based on levels of rhesus LCV DNA or RNA in the blood and lower rates of seroconversion. While animals that received VRP-gp350, VRP-EBNA-3A, and VRP-EBNA-3B had the next best level of protection from challenge and might have better protection from reactivation, than those receiving the other vaccine candidates, we could not test for protection against reactivation with the small number of animals in the current study. Although soluble gp350 induced the highest levels of antibody to gp350 and the best protection from acute infection, addition of potent EBV-specific T cell responses in combination with high levels of antibody might enhance the effectiveness of an EBV vaccine.
Although an ideal vaccine would protect from infection with EBV, a vaccine that reduces the EBV DNA load might also be useful. The EBV DNA load is a predictor for development of certain EBV-associated malignancies
In summary, our findings indicate that a subunit vaccine that induces primarily humoral, rather than cellular immunity can result in a low virus load in animals that develop breakthrough infection after challenge with wild-type virus. At 23 months after challenge, animals vaccinated with soluble gp350 that became infected with rhesus LCV had ≥100-fold lower levels of rhesus LCV DNA in PBMCs than those vaccinated with VRP-gp350, or the combination of VRP-gp350, VRP-EBNA-3A, and VRP-EBNA-3B. Rhesus LCV DNA was still lower in PBMCs from animals vaccinated with soluble gp350 at 34 months after challenge compared with animals that received PBS. Thus, antibodies to a viral glycoprotein before challenge likely alter the primary infection in such a way as to result in a lower viral load years later. While the largest EBV subunit vaccine study performed to date showed that soluble gp350 protected against infectious mononucleosis, breakthrough infection still occurred; however, the authors did not report on the level of EBV DNA in the blood after breakthrough infection
We thank Fred Wang (Brigham and Women's Hospital, Harvard Medical School) for advice regarding rhesus lymphocryptovirus, Peter Barry (University of California, Davis) for assistance with the animal protocol, and Yanmei Wang (National Institute of Allergy and Infectious Diseases) for help with real time PCR.