Research ArticleVaccines

Enhancing safety of cytomegalovirus-based vaccine vectors by engaging host intrinsic immunity

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Science Translational Medicine  17 Jul 2019:
Vol. 11, Issue 501, eaaw2603
DOI: 10.1126/scitranslmed.aaw2603
  • Fig. 1 RhCMV pp71 degrades DAXX and absence of pp71 results in in vitro growth deficiency.

    (A) TRFs or TRF-pp71 were untreated (−) or treated with DOX (+) (10 μg/ml) to induce RhCMV pp71 expression for 24 hours. Nuclear lysates were harvested and analyzed by immunoblot using anti-hemagglutinin (HA) Abs to detect epitope-tagged pp71. The cellular nuclear matrix protein p84 was analyzed as a loading control. The graph shows mean (+SD) with n = 2. (B) TRFs were infected with 68-1 or ΔRh110 (MOI = 3) or left uninfected, and cell lysates were harvested at the indicated time points, electrophoretically separated, and subjected to immunoblots with Abs to the indicated host and viral proteins. Each time point represents an independent infection. (C) TRFs were infected with 68-1 or either complemented or uncomplemented (−/−) ΔRh110/SIVgag at the indicated MOI. Supernatants were harvested at the indicated times and titered by TCID50 (median tissue culture infectious dose) on TRF-pp71. Average titers from two experimental and two technical replicates (+SD) are shown. (D) TRFs or TRF-pp71 were infected with 68-1, complemented ΔRh110/SIVgag, or uncomplemented ΔRh110 (ΔRh110−/−) at MOI = 0.001. Plaques were analyzed at 7 dpi using phase microscopy, and plaque size was measured using Adobe Photoshop. Individual plaque sizes, as well as average (±SD) from one of two experiments, are shown. The Kruskal-Wallis (KW) test was used to determine the significance of differences between the three groups [P = 0.0002, left; P = not significant (NS), right], with the Wilcoxon rank sum test used to perform pairwise analysis if KW P values were ≤0.05; brackets indicate pairwise comparisons with two-sided Wilcoxon P ≤ 0.05. P ≤ 0.05 is considered statistically significant.

  • Fig. 2 Rh110 (pp71)–deleted RhCMV retains T cell immunogenicity but is no longer shed in urine.

    (A) Frequencies of RhCMV-specific CD4+ and CD8+ T cell responses in PBMCs were determined at the indicated time points in two RMs inoculated with 107 PFU of ΔRh110 and three RMs given the same dose of 68-1 RhCMV. RhCMV IE1– and pp65a-specific T cells were determined by flow cytometric intracellular cytokine staining (ICS) after stimulation with mixes of consecutive, overlapping peptides comprising the RhCMV IE1 and pp65a proteins using intracellular expression of CD69 and either or both TNF-α and IFN-γ to define Ag-specific T cells. Response frequencies to IE1 and pp65a within the memory subset after background subtraction for each of the two ΔRh110-inoculated RMs (RM 2A and RM 2B) and the mean (+SEM) of these response frequencies for the three RMs given 68-1 RhCMV are shown. (B) Frequencies of RhCMV-specific T cell responses in bronchoalveolar lavages (BAL) were determined as in (A) in the same animals at the indicated time points. (C) Urine was isolated at the indicated dpi from RM 2A and RM 2B or one RM inoculated with 68-1. The presence of virus in cocultures was determined by immunoblot for RhCMV IE1 (see Materials and Methods).

  • Fig. 3 Durability, functional phenotype, and epitope targeting of SIV insert–specific T cell responses elicited by Rh110 (pp71)–deleted RhCMV vectors in naturally RhCMV-infected RMs.

    (A) Four naturally RhCMV-infected RMs were co-inoculated with 107 PFU of 68-1/SIVpol-5′ and the same dose of ΔRh110/SIVrtni, and flow cytometric ICS was used to follow the magnitude of the CD4+ and CD8+ T cell responses in peripheral blood to SIVpol and SIVrtn peptide mixes as described in Fig. 2. The mean + SEM of SIVpol- and SIVrtn-specific response frequencies within the memory CD4+ (left) and CD8+ (right) T cell populations are shown. (B) Boxplots compare the memory differentiation of the RhCMV-elicited CD4+ and CD8+ memory T cells in PBMCs [of the same RM shown in (A)] responding to SIVpol or SIVrtn with TNF-α and/or IFN-γ production at 630 dpi. Memory differentiation state was based on CD28 and CCR7 expression, delineating central memory (TCM), transitional effector memory (TTrEM), and effector memory (TEM), as designated. The Wilcoxon rank sum test was used to pairwise compare differences between the fraction of SIVpol- and SIVrtn-specific CD4+ and CD8+ T cells within each memory subset, with P = NS for all comparisons. (C) Boxplots compare the frequency of RhCMV-elicited CD4+ and CD8+ memory T cells in PBMCs of the same RM shown in (A) responding to SIVpol or SIVrtn peptides with TNF-α, IFN-γ, IL-2, and MIP1-β production, alone and in all combinations at 630 dpi. The Wilcoxon rank sum test was used to pairwise compare differences between the fraction of SIVpol- and SIVrtn-specific CD4+ and CD8+ T cells expressing one, two, three, or four cytokines, with P = NS for all comparisons. (D) SIVgag-specific CD8+ T cells in the peripheral blood of six ΔRh110/SIVgag-inoculated RMs were epitope-mapped using a flow cytometric ICS assay (CD69, TNF-α, and IFN-γ readout, as described above) to detect recognition of each consecutive, overlapping 15-mer peptide comprising the SIVgag protein. Peptides resulting in specific CD8+ T cell responses are indicated by a box, with the color of the box designating MHC restriction as determined by blocking with the anti–pan–MHC-I monoclonal Ab (mAb) W6/32, the MHC-E–blocking peptide VL9, and the MHC-II–blocking peptide CLIP, as previously described (52, 53). The blue and green arrowheads indicate the positions of previously identified MHC-II– and MHC-E–restricted SIVgag supertopes, respectively.

  • Fig. 4 Potency of Rh110 (pp71)–deleted RhCMV vectors in superinfection.

    (A) Two naturally RhCMV-infected RMs were inoculated with 107 PFU of ΔRh110/SIVrtni on day 0 and again on day 455 (for RM 4A) or day 133 (for RM 4B), the latter inoculation in combination with 107 PFU of ΔRh110/SIVenv. The panels show longitudinal analysis of the SIVrtn- and SIVenv-specific CD4+ and CD8+ T cell response frequencies among PBMCs determined by flow cytometric ICS (CD69, TNF-α, and IFN-γ readout) for each animal. (B) At time point 0, three groups (n = 3 per group) of naturally RhCMV-infected RMs were subcutaneously inoculated with the indicated dose (102, 104, and 106 PFU) of ΔRh110/SIVgag, complemented for pp71 by growing in TRF-pp71. The three RMs given 102 PFU did not manifest a detectable SIVgag-specific T cell response through 112 days of observation and were reinoculated with 103 PFU dose of the same vector. At the same time, all nine RMs were inoculated with 101 PFU of 68-1/SIVpol-5′. The figures show longitudinal analysis of the mean + SEM of SIVgag- and SIVpol-specific CD4+ and CD8+ T cell response frequencies among PBMCs, determined as described in (A). (C) Three naturally RhCMV-infected RMs were subcutaneously inoculated with 103 PFU of pp71-complemented ΔRh110/SIVenv, 103 PFU of ΔRh110/SIVgag, and 104 PFU ΔRh110/SIVrtni, with the latter two vectors grown in TRFs and thus not complemented for pp71. SIVenv-, SIVgag-, and SIVrtn-specific CD4+ and CD8+ T cell responses within the peripheral blood memory compartment were followed by flow cytometric ICS as described above (mean + SEM shown at each time point).

  • Fig. 5 Genetic stability of Rh110 (pp71)–deleted RhCMV vectors in the setting of superinfection.

    Two naturally RhCMV-infected RMs were each inoculated with 107 PFU of each dual insert–expressing ΔRh110/SIVenv/ΔRh19/SIVpol-5′ (A), ΔRh110/SIVenv/ΔRh107/SIVpol-5′ (B), or ΔRh110/SIVenv ΔRh192/SIVpol-5′ (C), and flow cytometric ICS was used to follow the SIVenv- and SIVpol-specific CD8+ T cell responses in peripheral blood, as described in Fig. 2. Dashed and solid lines each delineate the individual RM among the RM pairs given each vector. The relative position of the SIV antigens replacing endogenous genes in the viral genome is shown schematically below the graphs. CD4+ T cell responses from the same RM are shown in fig. S9. (D) Urine samples from the indicated time points after vector inoculation were analyzed for vector shedding by viral coculture, followed by Western blot (WB) analysis of SIVpol-5′ (top) or SIVenv (bottom) expression. Urine from RMs that previously received 68-1/SIVpol-5′ and 68-1/SIVenv vectors was included as a positive control.

  • Fig. 6 Lack of maternal-infant transmission of Rh110 (pp71)–deleted RhCMV vectors.

    (A) A naturally RhCMV+ pregnant dam was inoculated twice, as shown, with a panel of five 68-1 vectors (5 × 106 PFU each) expressing SIVenv, SIVgag, SIVrtni, SIVpol-3′ or SIVpol-5′. The dam gave birth to a healthy infant at 16 weeks after the first inoculation. The infant was cohoused with, and nursed from, the inoculated dam for 88 weeks, at which time the infant was weaned and cohoused with another, already naturally RhCMV+ juvenile RM. The mother, infant, and cohoused cagemate were followed for total SIV-specific CD4+ T cell responses (SIVgag + pol + rtn + env) in peripheral blood by flow cytometric ICS, with the response frequencies in the memory compartment shown. (B to F) Five naturally RhCMV-infected female RMs were inoculated with a panel of five ΔRh110 vectors (5 × 106 PFU each) expressing SIVenv, SIVgag, SIVrtn, SIVpol-3′, or SIVpol-5′ while nursing 17- to 28-week-old infants and were reinoculated 16 weeks later with the same vectors at the same dose. Vaccinated mothers and nursing infants were cohoused for a total of 48 weeks. Both mothers and infants were longitudinally followed for total SIV-specific CD4+ T cell responses by flow cytometric ICS. For (A) to (F), CD8+ T cell responses are shown in fig. S10.

  • Fig. 7 Deletion of the UL35 ortholog Rh59 from RhCMV results in growth deficiency in vitro while maintaining immunogenicity in vivo.

    (A) TRFs were infected with 68-1/SIVgag or ΔRh59/SIVgag at the indicated MOI. The supernatant was harvested at the indicated dpi and titered by TCID50 on TRFs. Average titers from two experimental and two technical replicates (+SD) are shown. (B) TRFs were infected with 68-1/SIVgag or ΔRh59/SIVgag at MOI = 0.01 or 0.001. Plaques were analyzed at 7 dpi using phase microscopy, and plaque size was measured using Adobe Photoshop. Individual plaque sizes and average (±SD) from one of three experiments are shown. Statistical significance was determined by the Wilcoxon rank sum test, with P ≤ 0.05 considered significant. (C) Two RMs were co-inoculated with 107 PFU of 68-1/SIVpol-5′ and ΔRh59/SIVgag, and the T cell responses to peptide mixes comprising SIVpol and SIVgag were longitudinally monitored in peripheral blood by flow cytometric ICS (CD69, TNF-α, and IFN-γ readout), with the response frequencies in the memory compartment shown for each RM (one designated by solid lines, the other by dashed lines).

  • Fig. 8 Comparison of the shedding and transmission upon leukocyte transfer of Rh110 (pp71)–deleted, Rh59 (UL35)–deleted, and 68-1 RhCMV vectors.

    (A) Four RMs were co-inoculated with 106 to 107 PFU of ΔRh59/SIVrtni, pentameric complex–repaired ΔRh110 68-1.2/SIVgag, ΔRh110/SIVenv, and 68-1/SIVpol-5′ at the designated time points, and the CD8+ T cell responses to peptide mixes comprising each of the SIV antigens were longitudinally monitored in peripheral blood by flow cytometric ICS (CD69, TNF-α, and IFN-γ readout), with the response frequencies in the memory compartment shown (see also fig. S13B). (B) Immunoblots of viral cocultures from urine samples obtained at the indicated days. Each of the SIV inserts carries a different epitope tag, allowing specific identification of each vector using tag-specific mAbs (see Materials and Methods). (C) Bone marrow and blood leukocytes from two of the RMs shown in (A) [1.9 × 107 bone marrow and 3.0 × 107 blood cells from RM 8A (donor 1); 0.8 × 107 bone marrow and 3.0 × 107 blood cells from RM 8B (donor 2); obtained at the indicated time point] were transferred to two naturally RhCMV+ (but vector naïve) RMs to test the ability of leukocyte transfer to transmit each vector to a new host. Vector infection of the new host was determined by longitudinal assessment of CD4+ and CD8+ T cell responses to each of the four different SIV inserts, as described in (A).

  • Table 1 Rh110 (pp71) deletion reduces dissemination of RhCMV vectors.

    (A) Three RhCMV-naïve RMs (T1A-1, T1A-2, and T1A-3) were co-inoculated with 107 PFU each of 68-1/SIVgag (left arm) and ΔRh110/SIVrtni (right arm). One RM each was necropsied at 14, 21, or 28 dpi, and viral genome copy numbers per 107 cell equivalents were determined in the indicated tissues using ultrasensitive nested qPCR specific for SIVgag (68-1) or SIVrtni (ΔRh110). (B) Three naturally RhCMV-infected RMs (T1B-1, T1B-2, and T1B-3) were co-inoculated with 107 PFU each of 68-1/SIVrtni (left arm) and ΔRh110/SIVgag (right arm). One RM each was necropsied at 14, 21, or 28 dpi, and viral genome copy numbers per 107 cell equivalents in the indicated tissues were determined using ultrasensitive nested qPCR specific for SIVgag (ΔRh110) or SIVrtni (68-1). (C) Two naturally RhCMV-infected RMs (T1C-1 and T1C-2) were co-inoculated with 107 PFU each of complemented ΔRh110 and uncomplemented ΔRh110 in different arms, with the SIVgag and SIVrtni inserts used to mark the complemented and uncomplemented vectors, respectively, in RM1 and the reverse in RM2. Both RMs were necropsied at 14 dpi, and viral genome copy numbers were determined using ultrasensitive nested qPCR specific for SIVgag versus SIVrtni. Normalized to 1 × 107 cell equivalents. WT, wild type; GI, gastrointestinal; LN, lymph node; PLN, parietal lymph node.

    A. Primary infection: 68-1 versus ΔRh110
    RM T1A-1RM T1A-2RM T1A-3
    Tissue type14 dpi21 dpi28 dpi
    68-1/SIVgagΔRh110/SIVrtni681/SIVgagΔRh110/
    SIVrtni
    68-1/SIVgagΔRh110/
    SIVrtni
    Skin injection site—right (ΔRh110)19,975,462<113,2272<1<1
    Skin injection site—left (WT)1,013,192,441314,453<1<1<1
    Axillary LN—right
    (ΔRh110 draining)
    4,547<1<1<17<1
    Axillary LN—left (WT draining)17,687,789<129,484<15,771<1
    PLN (except draining LN)265,298493<140<1
    Mesenteric LN<115<15<1
    GI tract3458<164<1
    Liver/gallbladder11<1<1<141<1
    Heart/lung/kidney/BAL66477<1<1<1
    BM/spleen/tonsil439,736<1<1<1
    Neuro/endocrine14<18<1<1<1
    Genitourinary tract1251414,075<117<1
    Salivary glands13,262<11,242<111<1
    PBMCs<13<1<1<1<1
    B. Superinfection: 68-1 versus ΔRh110
    RM T1B-1RM T1B-2RM T1B-3
    Tissue type14 dpi21 dpi28 dpi
    68-1/SIVrtniΔRh110/SIVgag68-1/SIVrtniΔRh110/
    SIVgag
    68-1/SIVrtniΔRh110/
    SIVgag
    Skin injection site—right (WT)47516,502<1376<1
    Skin injection site—left (ΔRh110)113547<1355<1
    Axillary LN—right
    (WT draining)
    117<17<1509<1
    Axillary LN—left
    (ΔRh110 draining)
    <1<1<1<1<1<1
    Peripheral LN1,434<141<12<1
    Mesenteric LN<111<1<1<1
    GI tract3<1<1<112<1
    Liver/gallbladder1<19<19<1
    Heart/lung/kidney/BAL5<1<1<18<1
    BM/spleen/tonsil155<1<1<1<1
    Neuro/endocrine<1<13<13<1
    Genitourinary tract3<14<11,047<1
    Salivary gland231<1<1<1<1
    C. ΔRh110: Complemented versus uncomplemented
    RM T1C-1←14 dpi→RM T1C-2
    Tissue typeUncomplemented
    ΔRh110/SIVrtni
    Complemented
    ΔRh110/SIVgag
    Tissue typeUncomplemented
    ΔRh110/SIVgag
    Complemented
    ΔRh110/SIVrtni
    Skin injection site—right
    (complemented)
    <1<1Skin injection site—right
    (uncomplemented)
    <1<1
    Skin injection site—left
    (uncomplemented)
    13<1Skin injection site—left
    (complemented)
    <1<1
    Axillary LN—right
    (complemented draining)
    <1<1Axillary LN—right
    (uncomplemented
    draining)
    <1<1
    Axillary LN—left
    (uncomplemented draining)
    17<1Axillary LN—left
    (complemented
    draining)
    <1<1
    PLN (except draining LN)<1<1PLN (except draining
    LN)
    <1<1
    Mesenteric LN<1<1Mesenteric LN<1<1
    GI tract<1<1GI tract<1<1
    Liver/gallbladder<1<1Liver/gallbladder<1<1
    Heart/lung/kidney/BAL<1<1Heart/lung/kidney/BAL<1<1
    BM/spleen/tonsil<1<1BM/spleen/tonsil<1<1
    Neuro/endocrine<1<1Neuro/endocrine<1<1
    Genitourinary tract5<1Genitourinary tract<12
    Salivary glands<1<1Salivary glands<1<1
    PBMCs<1<1PBMCs<1<1
  • Table 2 Rh59 (UL35) deletion reduces dissemination of 68-1 RhCMV vectors.

    Three naturally RhCMV-infected RMs (T2-1, T2-2, and T2-3) were co-inoculated with 107 PFU each of 68-1/SIVrtni (left arm) and ΔRh59/SIVgag (right arm). One RM was necropsied at 14, 21, or 28 dpi, and viral genome copy numbers per 107 cell equivalents in the indicated tissues were determined using ultrasensitive nested qPCR specific for SIVrtni (68-1) or SIVgag (ΔRh59). Normalized to 1 × 107 cell equivalents.

    Super infection: 68-1 versus ΔRh59
    RM T2-1RM T2-2RM T2-3
    Tissue type14 dpi21 dpi28 dpi
    68-1/SIVrtniΔRh59/SIVgag68-1/SIVrtniΔRh59/SIVgag68-1/SIVrtniΔRh59/SIVgag
    Skin injection site—
    right (ΔRh59)
    12<1193<136,1107
    Skin injection site—
    left (68–1)
    541<123,793<14,480,601<1
    Axillary LN3<113<12,5157
    Peripheral LN<1<1<1<118<1
    Liver/gallbladder<1<1<1<12<1
    Heart/lung/kidney/
    BAL
    2,933<13<16<1
    Bone marrow/spleen/
    tonsil
    6<1<1<18<1
    Neuro/endocrine3<1<1<141
    Genitourinary tract<1<13<19<1
    Salivary gland2<14<13<1

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/501/eaaw2603/DC1

    Materials and Methods

    Fig. S1. Redistribution of corepressors by RhCMV pp71.

    Fig. S2. Description and characterization of pp71-deleted RhCMV vectors.

    Fig. S3. Summary of recombinant RhCMV/SIV vectors used in vivo.

    Fig. S4. Tissue distribution of SIV insert–specific CD4+ and CD8+ T cell responses elicited by ΔRh110 versus 68-1 vectors.

    Fig. S5. Urine shedding of ΔRh110 versus 68-1 vectors.

    Fig. S6. T cell responses in lung airspace (BAL cells) upon super-infection with pp71-deleted RhCMV vectors.

    Fig. S7. Boosting of RhCMV Ab responses by ΔRh110 (Δpp71) RhCMV vectors.

    Fig. S8. Description and characterization of dual insert–expressing, pp71-deleted RhCMV vectors.

    Fig. S9. CD4+ T cell responses to dual antigen insert–expressing, pp71-deleted RhCMV vectors.

    Fig. S10. Lack of maternal-infant transmission of pp71-deleted vectors.

    Fig. S11. Description and characterization of Rh59 (UL35)–deleted RhCMV used in this study.

    Fig. S12. SIV antigen–specific CD4+ and CD8+ T cell responses elicited by ΔRh59 versus 68-1 RhCMV vectors in tissue sites.

    Fig. S13. T cell responses to SIV antigens expressed by ΔRh110 and ΔRh59 vectors.

    Data file S1. Primary data.

    References (7681)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Redistribution of corepressors by RhCMV pp71.
    • Fig. S2. Description and characterization of pp71-deleted RhCMV vectors.
    • Fig. S3. Summary of recombinant RhCMV/SIV vectors used in vivo.
    • Fig. S4. Tissue distribution of SIV insert–specific CD4+ and CD8+ T cell responses elicited by ΔRh110 versus 68-1 vectors.
    • Fig. S5. Urine shedding of ΔRh110 versus 68-1 vectors.
    • Fig. S6. T cell responses in lung airspace (BAL cells) upon super-infection with pp71-deleted RhCMV vectors.
    • Fig. S7. Boosting of RhCMV Ab responses by ΔRh110 (Δpp71) RhCMV vectors.
    • Fig. S8. Description and characterization of dual insert–expressing, pp71-deleted RhCMV vectors.
    • Fig. S9. CD4+ T cell responses to dual antigen insert–expressing, pp71-deleted RhCMV vectors.
    • Fig. S10. Lack of maternal-infant transmission of pp71-deleted vectors.
    • Fig. S11. Description and characterization of Rh59 (UL35)–deleted RhCMV used in this study.
    • Fig. S12. SIV antigen–specific CD4+ and CD8+ T cell responses elicited by ΔRh59 versus 68-1 RhCMV vectors in tissue sites.
    • Fig. S13. T cell responses to SIV antigens expressed by ΔRh110 and ΔRh59 vectors.
    • References (7681)

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    Other Supplementary Material for this manuscript includes the following:

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