Christopher S. Morello

Genome-Wide Analysis Reveals a Highly Diverse CD8 T Cell Response to Murine Cytomegalovirus

Human CMV establishes a lifelong latent infection in the majority of people worldwide. Although most infections are asymptomatic, immunocompetent hosts devote an extraordinary amount of immune resources to virus control. To increase our understanding of CMV immunobiology in an animal model, we used a genomic approach to comprehensively map the C57BL/6 CD8 T cell response to murine CMV (MCMV). Responses to 27 viral proteins were detectable directly ex vivo, the most diverse CD8 T cell response yet described within an individual animal. Twenty-four peptide epitopes were mapped from 18 Ags, which together account for most of the MCMV-specific response. Most Ags were from genes expressed at early times, after viral genes that interfere with Ag presentation are expressed, consistent with the hypothesis that the CD8 T cell response to MCMV is largely driven by cross-presented Ag. Titration of peptide epitopes in a direct ex vivo intracellular cytokine staining assay revealed a wide range of functional avidities, with no obvious correlation between functional avidity and the strength of the response. The immunodominance hierarchy varied only slightly between mice and between experiments. However, H-2b-expressing mice with different genetic backgrounds responded preferentially to different epitopes, indicating that non-MHC-encoded factors contribute to immunodominance in the CD8 T cell response to MCMV.

Suppression of Murine Cytomegalovirus (MCMV) Replication with a DNA Vaccine Encoding MCMV M84 (a Homolog of Human Cytomegalovirus pp65)

ABSTRACT The cytotoxic T-lymphocyte (CTL) response against the murine cytomegalovirus (MCMV) immediate-early gene 1 (IE1) 89-kDa phosphoprotein pp89 plays a major role in protecting BALB/c mice against the lethal effects of the viral infection. CTL populations specific to MCMV early-phase and structural antigens are also generated during infection, but the identities of these antigens and their relative contributions to overall immunity against MCMV are not known. We previously demonstrated that DNA vaccination with a pp89-expressing plasmid effectively generated a CTL response and conferred protection against infection (J. C. Gonzalez Armas, C. S. Morello, L. D. Cranmer, and D. H. Spector, J. Virol. 70:7921–7928, 1996). In this report, we have sought (i) to identify other viral antigens that contribute to immunity against MCMV and (ii) to determine whether the protective response is haplotype specific. DNA immunization was used to test the protective efficacies of plasmids encoding MCMV homologs of human cytomegalovirus (HCMV) tegument (M32, M48, M56, M82, M83, M69, and M99), capsid (M85 and M86), and nonstructural antigens (IE1-pp89 and M84). BALB/c (H-2d ) and C3H/HeN (H-2k ) mice were immunized by intradermal injection of either single plasmids or cocktails of up to four expression plasmids and then challenged with sublethal doses of virulent MCMV administered intraperitoneally. In this way, we identified a new viral gene product, M84, that conferred protection against viral replication in the spleens of BALB/c mice. M84 is expressed early in the infection and encodes a nonstructural protein that shares significant amino acid homology with the HCMV UL83-pp65 tegument protein, a major target of protective CTLs in humans. Specificity of the immune response to the M84 protein was confirmed by showing that immunization with pp89 DNA, but not M84 DNA, protected mice against subsequent infection with an MCMV deletion mutant lacking the M84 gene. The other MCMV genes tested did not generate a protective response even when mice were immunized with vaccinia viruses expressing the viral proteins. However, the M84 plasmid was protective when injected in combination with nonprotective plasmids, and coimmunization of BALB/c mice with pp89 and M84 provided a synergistic level of protection in the spleen. Viral titers in the salivary glands were also reduced, but not to the same extent as observed in the spleen, and the decrease was seen only when the BALB/c mice were immunized with pp89 plus M84 or with pp89 alone. The experiments with the C3H/HeN mice showed that the immunity conferred by DNA vaccination was haplotype dependent. In this strain of mice, only pp89 elicited a protective response as measured by a reduction in spleen titer. These results suggest that DNA immunization with the appropriate combination of CMV genes may provide a strategy for improving vaccine efficacy.

Development of a Vaccine against Murine Cytomegalovirus (MCMV), Consisting of Plasmid DNA and Formalin-Inactivated MCMV, That Provides Long-Term, Complete Protection against Viral Replication

ABSTRACT We previously demonstrated that immunization of mice with plasmid DNAs (pDNAs) expressing the murine cytomegalovirus (MCMV) genes IE1-pp89 and M84 provided synergistic protection against sublethal viral challenge, while immunization with plasmids expressing putative virion proteins provided no or inconsistent protection. In this report, we sought to augment protection by increasing the breadth of the immune response. We identified another MCMV gene (m04 encoding gp34) that provided strong and consistent protection against viral replication in the spleen. We also found that immunization with a DNA pool containing 10 MCMV genes that individually were nonprotective elicited reproducible protection against low to intermediate doses of challenge virus. Moreover, inclusion of these plasmids into a mixture with gp34, pp89, and M84 DNAs provided even greater protection than did coimmunization with pp89 and M84. The highest level of protection was achieved by immunization of mice with the pool of 13 pDNAs, followed by formalin-inactivated MCMV (FI-MCMV). Immunization with FI-MCMV elicited neutralizing antibodies against salivary gland-derived MCMV, and of greatest importance, mice immunized with both the combined pDNA pool and FI-MCMV had undetectable levels of virus in the spleen and salivary glands after challenge. Intracellular cytokine staining of splenocytes from pDNA- and FI-MCMV-immunized mice showed that pDNA immunization elicited high levels of pp89- and M83-specific CD8+ T cells, whereas both pDNA and FI-MCMV immunizations generated strong CD8+-T-cell responses against virion-associated antigens. Taken together, these results show that immunization with pDNA and inactivated virus provides strong antibody and cell-mediated immunity against CMV infection.

Recombinant antibodies encoded by IGHV1-69 react with pUL32, a phosphoprotein of cytomegalovirus and B-cell superantigen

Leukemia cells from patients with chronic lymphocytic leukemia (CLL) express a highly restricted immunoglobulin heavy variable chain (IGHV) repertoire, suggesting that a limited set of antigens reacts with leukemic cells. Here, we evaluated the reactivity of a panel of different CLL recombinant antibodies (rAbs) encoded by the most commonly expressed IGHV genes with a panel of selected viral and bacterial pathogens. Six different CLL rAbs encoded by IGHV1-69 or IGHV3-21, but not a CLL rAb encoded by IGHV4-39 genes, reacted with a single protein of human cytomegalovirus (CMV). The CMV protein was identified as the large structural phosphoprotein pUL32. In contrast, none of the CLL rAbs bound to any other structure of CMV, adenovirus serotype 2, Salmonella enterica serovar Typhimurium, or of cells used for propagation of these microorganisms. Monoclonal antibodies or humanized rAbs of irrelevant specificity to pUL32 did not react with any of the proteins present in the different lysates. Still, rAbs encoded by a germ line IGHV1-69 51p1 allele from CMV-seropositive and -negative adults also reacted with pUL32. The observed reactivity of multiple different CLL rAbs and natural antibodies from CMV-seronegative adults with pUL32 is consistent with the properties of a superantigen.

Immunization with Herpes Simplex Virus 2 (HSV-2) Genes plus Inactivated HSV-2 Is Highly Protective against Acute and Recurrent HSV-2 Disease

ABSTRACT To date, no vaccine that is safe and effective against herpes simplex virus 2 (HSV-2) disease has been licensed. In this study, we evaluated a DNA prime-formalin-inactivated-HSV-2 (FI-HSV2) boost vaccine approach in the guinea pig model of acute and recurrent HSV-2 genital disease. Five groups of guinea pigs were immunized and intravaginally challenged with HSV-2. Two groups were primed with plasmid DNAs encoding the secreted form of glycoprotein D2 (gD2t) together with two genes required for viral replication, either the helicase (UL5) and DNA polymerase (UL30) genes or the single-stranded DNA binding protein (UL29) and primase (UL52) genes. Both DNA-primed groups were boosted with FI-HSV2 formulated with monophosphoryl lipid A (MPL) and alum adjuvants. Two additional groups were primed with the empty backbone plasmid DNA (pVAX). These two groups were boosted with MPL and alum (MPL-alum) together with either formalin-inactivated mock HSV-2 (FI-Mock) or with FI-HSV2. The final group was immunized with gD2t protein in MPL-alum. After challenge, 0/9 animals in the group primed with UL5, UL30, and gD2t DNAs and all 10 animals in the mock-immunized control group (pVAX-FI-Mock) developed primary lesions. All mock controls developed recurrent lesions through day 100 postchallenge. Only 1 guinea pig in the group primed with pVAX DNA and boosted with FI-HSV2 (pVAX-FI-HSV2 group) and 2 guinea pigs in the group primed with UL5, UL30, and gD2t DNAs and boosted with FI-HSV2 (UL5, UL30, gD2t DNA-FI-HSV2 group) developed recurrent lesions. Strikingly, the UL5, UL30, gD2t DNA-FI-HSV2 group showed a 97% reduction in recurrent lesion days compared with the mock controls, had the highest reduction in days with recurrent disease, and contained the lowest mean HSV-2 DNA load in the dorsal root ganglia.

Systemic Priming-Boosting Immunization with a Trivalent Plasmid DNA and Inactivated Murine Cytomegalovirus (MCMV) Vaccine Provides Long-Term Protection against Viral Replication following Systemic or Mucosal MCMV Challenge

ABSTRACT We previously demonstrated that vaccination of BALB/c mice with a pool of 13 plasmid DNAs (pDNAs) expressing murine cytomegalovirus (MCMV) genes followed by formalin-inactivated MCMV (FI-MCMV) resulted in complete protection against viral replication in the spleen and salivary glands following sublethal intraperitoneal (i.p.) challenge. Here, we found that following intranasal (i.n.) challenge, titers of virus in the lungs of the immunized mice were reduced approximately 1,000-fold relative to those for mock-immunized controls. We next sought to extend these results and to determine whether similar protection levels could be achieved by priming with a pool of three pDNAs containing three key plasmids (IE1, M84, and gB). We found that the three-pDNA priming elicited IE1- and M84-p65-specific CD8+ T lymphocytes and, following FI-MCMV boost, high levels of virion-specific immunoglobulin G (IgG) and virus-neutralizing antibodies. When mice were i.n. challenged 4 months after the last boost, titers of virus in the lungs of immunized mice were reduced 1,000- to 2,000-fold from those for controls during the peak of viral replication. Additionally, titers of virus were either at or below the detection limits for the salivary glands, liver, and spleen of the majority of the immunized mice. Following sublethal i.p. challenge, virus was undetectable in all of the above target organs of the immunized mice. Virion-specific IgA in the lungs was consistently detected by day 6 post-i.n. challenge for the immunized mice and by day 14 for controls. These results demonstrate the immunity and high levels of protection of the priming-boosting vaccination against both systemic and mucosal challenge.

25-Hydroxycholesterol Activates the Integrated Stress Response to Reprogram Transcription and Translation in Macrophages

Background: Interferons and viral infections stimulate the production of 25-hydroxycholesterol. Results: 25-Hydroxycholesterol significantly alters cholesterol ester and sphingolipid levels and activates the integrated stress response. Conclusion: 25-Hydroxycholesterol activates the GCN2/eIF2α/ATF4 integrated stress response likely by causing cysteine depletion and/or by generating oxidative stress. Significance: Altering important membrane lipids and activating the integrated stress response may contribute to the antiviral activity of 25-hydroxycholesterol. 25-Hydroxycholesterol (25OHC) is an enzymatically derived oxidation product of cholesterol that modulates lipid metabolism and immunity. 25OHC is synthesized in response to interferons and exerts broad antiviral activity by as yet poorly characterized mechanisms. To gain further insights into the basis for antiviral activity, we evaluated time-dependent responses of the macrophage lipidome and transcriptome to 25OHC treatment. In addition to altering specific aspects of cholesterol and sphingolipid metabolism, we found that 25OHC activates integrated stress response (ISR) genes and reprograms protein translation. Effects of 25OHC on ISR gene expression were independent of liver X receptors and sterol-response element-binding proteins and instead primarily resulted from activation of the GCN2/eIF2α/ATF4 branch of the ISR pathway. These studies reveal that 25OHC activates the integrated stress response, which may contribute to its antiviral activity.

DNA immunization using highly conserved murine cytomegalovirus genes encoding homologs of human cytomegalovirus UL54 (DNA polymerase) and UL105 (helicase) elicits strong CD8 T-cell responses and is protective against systemic challenge.

ABSTRACT Human cytomegalovirus (HCMV) establishes a lifelong infection with the potential for reinfection or viral transmission even in the presence of strong and diverse CD8 T-lymphocyte responses. This suggests that the CMVs skew the host T-cell response in order to favor viral persistence. In this study, we hypothesized that the essential, nonstructural proteins that are highly conserved among the CMVs may represent a novel class of T-cell targets for vaccine-mediated protection due to their requirements for expression and sequence stability, but that the observed subdominance of these antigens in the CMV-infected host results from the virus limiting the T-cell responses to otherwise-protective specificities. We found that DNA immunization of mice with the murine CMV (MCMV) homologs of HCMV DNA polymerase (M54) or helicase (M105) was protective against virus replication in the spleen following systemic challenge, with the protection level elicited by the M54 DNA being comparable to that of DNA expressing the immunodominant IE1 (pp89). Intracellular gamma interferon staining of CD8 T cells from mice immunized with either the M54 or M105 DNAs showed strong primary responses that recalled rapidly after viral challenge. M54- and M105-specific CD8 T cells were detected after the primary MCMV infection, but their levels were not consistently above the background level. The conserved, essential proteins of the CMVs thus represent a novel class of CD8 T-cell targets that may contribute to a successful HCMV vaccine strategy.

Inactivated HSV-2 in MPL/Alum Adjuvant Provides Nearly Complete Protection Against Genital Infection and Shedding Following Long Term Challenge and Rechallenge

Herpes Simplex Virus Type 2 (HSV-2) infection can result in life-long recurrent genital disease, asymptomatic virus shedding, and transmission. No vaccine to date has shown significant protection clinically. Here, we used a mouse model of genital HSV-2 infection to test the efficacy of a vaccine consisting of whole, formalin-inactivated HSV-2 (FI-HSV2) formulated with monophosphoryl lipid A (MPL) and alum adjuvants. Vaccine components were administered alone or as a prime-boost immunization together with DNA vaccines encoding a truncated glycoprotein D2 (gD2t) and two conserved HSV-2 genes necessary for virus replication, UL5 (DNA helicase) and UL30 (DNA polymerase). Our results show: (1) compared with mock immunized controls, mice immunized with FI-HSV2 plus MPL/alum consistently showed protection against disease burden and total viral shedding while the mice immunized with gD2t protein with MPL/alum did not; (2) protection against genital disease and viral replication correlated with the type of boost in a prime-boost immunization with little advantage afforded by a DNA prime; (3) intramuscular (i.m.) immunization with FI-HSV2 in MPL/Alhydrogel adjuvant provided nearly complete protection against vaginal HSV-2 shedding after a lethal intravaginal (i.vag.) short-term challenge and long-term rechallenge; (4) single formulation immunization with DNA vaccines, FI-HSV2, and MPL in an aluminum phosphate (Adju-Phos) adjuvant did not increase protection relative to FI-HSV2/MPL/Adju-Phos alone; and (5) addition of MPL/alum to the FI-HSV2 was required for optimal protection against disease, viral replication, and latent virus load in the dorsal root ganglia (DRG). Most notably, an optimized vaccine formulation of FI-HSV2 MPL/Alhydrogel given i.m. completely protected against detectable vaginal HSV-2 shedding in the majority of animals and HSV-2 latent DNA in the DRG of all animals.

Development of Cell Lines That Provide Tightly Controlled Temporal Translation of the Human Cytomegalovirus IE2 Proteins for Complementation and Functional Analyses of Growth-Impaired and Nonviable IE2 Mutant Viruses

ABSTRACT The human cytomegalovirus (HCMV) IE2 86 protein is essential for viral replication. Two other proteins, IE2 60 and IE2 40, which arise from the C-terminal half of IE2 86, are important for later stages of the infection. Functional analyses of IE2 86 in the context of the infection have utilized bacterial artificial chromosomes as vectors to generate mutant viruses. One limitation is that many mutations result in debilitated or nonviable viruses. Here, we describe a novel system that allows tightly controlled temporal expression of the IE2 proteins and provides complementation of both growth-impaired and nonviable IE2 mutant viruses. The strategy involves creation of cell lines with separate lentiviruses expressing a bicistronic RNA with a selectable marker as the first open reading frame (ORF) and IE2 86, IE2 60, or IE2 40 as the second ORF. Induction of expression of the IE2 proteins occurs only following DNA recombination events mediated by Cre and FLP recombinases that delete the first ORF. HCMV encodes Cre and FLP, which are expressed at immediate-early (for IE2 86) and early-late (for IE2 40 and IE2 60) times, respectively. We show that the presence of full-length IE2 86 alone provides some complementation for virus production, but the correct temporal expression of IE2 86 and IE2 40 together has the most beneficial effect for early-late gene expression and synthesis of infectious virus. This approach for inducible protein translation can be used for complementation of other mutations as well as controlled expression of toxic cellular and microbial proteins.