A heterologous DNA prime/protein boost immunization strategy for rhesus cytomegalovirus

Abstract

A previous study in nonhuman primates demonstrated that genetic immunization against the rhesus cytomegalovirus phosphoprotein 65-2 (pp65-2) and glycoprotein B (gB) antigens both stimulated antigen-specific antibodies and CD8 T cell responses, and significantly reduced plasma viral loads following intravenous challenge with RhCMV. It was also noted in this study that weak CD4 T cell and neutralizing antibody responses were generated by DNA alone. To broaden the type of immune responses, a DNA prime/protein boost strategy was used in seronegative macaques, consisting of four DNA immunizations against pp65-2, gB, and immediate-early 1 (IE1), followed by two boosts with formalin-inactivated RhCMV virions. This heterologous prime/boost strategy elicited robust antigen-specific CD4 and CD8 T cell responses in addition to biologically relevant neutralizing antibody titers. Animals were challenged with RhCMV delivered into four sites via a subcutaneous route. Skin biopsies of one of the inoculation sites 7 days post challenge revealed marked differences in the level of RhCMV replication between the vaccinated and control monkeys. Whereas the inoculation site in the controls was noted for a prominent inflammatory response and numerous cytomegalic, antigen-positive (IE1) cells, the inoculation site in the vaccinees was characterized by an absence of inflammation and antigen-positive cells. All five vaccinees developed robust recall responses to viral antigens, and four of them exhibited long-term viral immune responses consistent with effective control of viral expression and replication. These results demonstrate that a heterologous DNA prime/protein boost strategy greatly expands the breadth of antiviral immune responses and greatly reduces the level of viral replication at the primary site of challenge infection.

Vaccine-Induced Control of Viral Shedding following Rhesus Cytomegalovirus Challenge in Rhesus Macaques

“…Animals were divided into four vaccine treatment groups as shown in Table 1: group 1 (n ϭ 6), mock-immunized controls; group 2 (n ϭ 6), animals immunized against RhCMV gB; group 3 (n ϭ 6), animals immunized against RhCMV gB, pp65-2, and IE1; and group 4 (n ϭ 3), animals immunized against RhCMV gB, pp65-2, and IE1 and boosted with formalin-inactivated RhCMV (FI-RhCMV) virions. Although group 4 was comprised of a smaller number of monkeys than the other groups (n ϭ 3 versus n ϭ 6), the goal was to provide a comparison of viral outcomes after challenge in this study with our previous study using RhCMV 68-1 as the challenge virus (2). The backbone pND expression vector and the expression plasmids for RhCMV pp65-2 (pND/65-2), the transmembrane deletion version of gB (pND/gB⌬TM), and RhCMV IE1 (exons 2 to 4) have been described previously (2,27,46,49).…”

“…Although group 4 was comprised of a smaller number of monkeys than the other groups (n ϭ 3 versus n ϭ 6), the goal was to provide a comparison of viral outcomes after challenge in this study with our previous study using RhCMV 68-1 as the challenge virus (2). The backbone pND expression vector and the expression plasmids for RhCMV pp65-2 (pND/65-2), the transmembrane deletion version of gB (pND/gB⌬TM), and RhCMV IE1 (exons 2 to 4) have been described previously (2,27,46,49). All expression plasmids were purified from bacteria using Endo-free plasmid extraction kits (Qiagen).…”

“…DNA was resuspended in phosphate-buffered saline (PBS) buffer (Invitrogen) at a concentration of 1 mg/ml and stored at Ϫ20°C. FI-RhCMV was prepared as previously described (2). Briefly, 22 mg of pelleted RhCMV virions (ϳ6 ϫ 10 9 PFU total) were formalin inactivated and resuspended at a concentration of 550 g/ml.…”

“…and intradermal (i.d.) injections as previously described (2,27,46). Modified vaccinia virus Ankara (MVA) vaccine vectors expressing RhCMV gB, pp65, and IE were constructed, purified, and expanded into amounts suitable for rhesus monkey studies as previously described (48).…”

“…Group 4 monkeys were boosted i.m. at weeks 6 and 12 with 100 g of FI-RhCMV adjuvanted with Montanide ISA 720 (Seppic Inc., Fairfield, NJ), as previously described (2).…”