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.

Fig. 1

Schedules for DNA vaccination and RhCMV inoculation. Animals were vaccinated intramuscularly (IM) and intradermally (ID) four times at weeks 0, 4, 8, and 12 with separate expression plasmids for gBΔ/pp65-2/ IE1 followed by two IM vaccinations at weeks 16 and 20 with formalin-inactivated RhCMV (FI-RhCMV) formulated in the Montanide ISA 720 adjuvant. Control animals were mock-vaccinated at the same time points with an “empty” pND vector (DNA) followed by two vaccinations with saline formulated in Montanide ISA 720. All animals were challenged with RhCMV strain 68-1 by subcutaneously (SC) inoculating the animals at four sites with a total of 2 × 10⁶ PFU of virus. Skin biopsies (Bx's) were obtained from one site at week +1 (relative to the time of challenge) and a second site at +10 days (not shown). Axillary lymph node (LN) biopsies were obtained at weeks +1 and +2. Blood draws and oral swabs were obtained throughout the 22 weeks of observation post challenge (not shown).

Fig. 2

A RhCMV seronegative macaque was immunized twice with FI-RhCMV (336 μg of total protein per immunization) at weeks 0 and 8. RhCMV-binding antibody responses were quantified by ELISA and are presented as Absorbency (at 450 nm). The threshold for a sample to be considered positive is indicated by the dotted line. The slight rise in absorbency at weeks 28–30 reflect the normal fluctuations in antibody responses that would be expected with weekly sampling.

Fig. 3

Antibody (Ab) responses to RhCMV gBΔTM post DNA ((A) 1:100 dilution of plasma) and FI-RhCMV ((B) 1:800 dilution), and to pp65-2 post FI-RhCMV ((C) 1:800 dilution). Arrows indicate times of immunization (in addition to week 0 in Fig. 1A), and dashed lines in (A) represent responses for two of the control animals immunized with the “empty” pND vector. The 50% neutralizing (NT₅₀) Ab titers are shown (D) beginning at Week 22 (2 weeks after the second immunization with FI-RhCMV).

Fig. 4

CD4⁺ (A and C) and CD8⁺ (B and D) T cell vaccine responses for TNF-α (A and B) and IFNγ (C and D) using overlapping peptide pools for RhCMV pp65-2 (RhCMV pp65) and IE1, and a lysate prepared from RhCMV-infected cells. The IE1 peptide pool was not available at the first assay timepoint 14 days after the last DNA immunization (d14 post DNA). The other timepoints are presented relative to the time of the first or second immunization with FI-RhCMV (#1 and #2, respectively). d28 post FI-RhCMV #1 and #2 represent the times for the second immunization with FI-RhCMV and RhCMV challenge (respectively).

Fig. 5

(A) Histological sections from one of the inoculations sites stained with hematoxylin and eosin (H and E). The representative sections from four animals from the control (top row) and vaccinated (bottom) groups obtained on either day 6 (D6) or 7 (D7) post inoculation are shown. Magnifications (10× or 20×) are indicated. HF: hair follicle. (B) Immunohistochemical staining for RhCMV IE1 in four of the five control and vaccinated animals, including the fifth animal of each group whose H and E section is not presented in (A). Antigen-positive cells are noted for brown-stained nuclei. Non-specific staining (NS) in some of the hair follicles is indicated.

Fig. 6

Post RhCMV challenge NT₅₀ and RhCMV-binding antibody responses are presented for the vaccinated (red) and control (blue) animals. The Relative Antibody Units for the RhCMV-binding Ab responses were based on serial dilutions of a plasma sample from a naturally RhCMV-infected macaque. An arbitrary value of 1 was assigned to that optical density (450 nm) for the serial dilutions that was above background and that was within the linear range of the assay. The Relative Antibody Units for the plasma samples from the control and vaccinated monkeys were derived from the ratio of the optical densities for those samples relative to the minimally positive optical density for the serial dilutions. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 7

Western blot analysis of IE1-specific antibody responses post vaccination and RhCMV challenge. A longitudinal analysis of anti-IE1 antibody responses post vaccination and challenge in the vaccinees (left half) and post RhCMV challenge in the controls (right half) is shown. Lane 1: week 0; lane 2: week 12 post priming immunization; lane 3: week 14; lane 4: week 16; lane 5: week 18; lane 6: week 20; lane 7: week 22; lanes 8 and 16: week 24 (relative to the time of priming immunization)/week +0 (relative to the time of RhCMV challenge); lanes 9 and 17: week +1 (relative to the time of RhCMV challenge); lanes 10 and 18: week +2; lanes 12 and 19: week +3; lanes 13 and 20: week +4; lanes 14 and 21: week +6; lanes 14 and 22: week +8; lanes 15 and 23: week +10. D: DNA immunizations at weeks 0 and 12. P: FI-RhCMV immunizations at weeks 16 and 20. R: RhCMV challenge at week 24 (relative to the time of the priming immunizations).

Fig. 8

Peripheral RhCMV-specific CD4⁺ responses (TNF-α, IFNγ, and IL-2) in the control and vaccine groups post RhCMV challenge.

Fig. 9

Peripheral RhCMV-specific CD8⁺ responses (TNF-α, IFNγ, and IL-2) in the control and vaccine groups post RhCMV challenge.

Fig. 10

RhCMV-specific CD8⁺ responses (TNF-α, IFNγ, and IL-2) in the axillary lymph node of the control and vaccine groups post RhCMV challenge.