Project 4

Development of vaccine strategies to prevent congenital cytomegalovirus infection

Human cytomegalovirus (HCMV) is the most common infectious cause of permanent birth defects worldwide, often leading to cognitive and auditory abnormalities and in rare cases even to multi-system organ failure and death. Persistent newborn medical conditions in the United States are more frequently associated with congenital HCMV infection than with other well-known childhood diseases such as trisomy 21, spina bifida, or fetal alcohol syndrome. In addition, HCMV is a frequent cause of disease in transplant recipients. As a consequence of the public health burden and absence of effective treatment options, governmental and academic authorities assigned the development of a HCMV vaccine of highest priority.
 
Neutralizing antibodies (NAb) that block viral glycoprotein complex (gC)-mediated entry into host cells are thought to be essential to prevent or control congenital HCMV infection. For many decades, HCMV subunit vaccine research has primarily focused on stimulation of NAb targeting the major essential envelope glycoprotein gB, culminating in the encouraging findings obtained with recombinant gB admixed in adjuvant MF59. In phase II clinical trials, gB/MF59 has been shown to reduce viremia and need for antiviral therapy in transplant recipients and to provide moderate efficacy of 38-50% to prevent primary infection in women of childbearing age. These findings have spurred interest to improve vaccine-mediated induction of NAb responses as an approach to achieve protection efficacy beyond that observed with gB/MF59.
 
Over the past decade it has been recognized that HCMV infection of fibroblasts (FB) and epithelial/endothelial cells (EC) occurs by distinct routes of entry that are blocked by neutralizing antibodies (NAb) of varying potency and cell-type specificity. NAb against the major essential envelope glycoprotein complexes (gC) gM/gN, gB, or gH/gL can inhibit both FB and EC entry. In contrast to FB entry, HCMV infection of EC requires a pentameric gC (PC) formed by gH/gL, UL128, UL130, and UL131A. NAb predominantly targeting conformational epitopes of the PC are unable to prevent FB entry, but they are significantly more potent than NAb to the major gC to block EC entry. Based on these findings, many investigators have developed preclinical PC based vaccine strategies to elicit EC specific NAb that exceed those induced by gB.
 
We developed a vaccine concept based on the delivery of a membrane-tethered PC by Modified Vaccinia virus Ankara (MVA), a widely used and safe poxviral vector that has been tested in over 120000 people. This vector stimulated high titer and durable EC-specific NAb in mice and rhesus monkeys (RM) that were dramatically higher than those induced by MVA expressing only subunit subsets of the PC (UL128/130/131A or gH/gL) or gB. In addition, MVA-PC and MVA expressing gH/gL or gB had similar ability to induce NAb blocking HCMV infection of FB, showing that the PC has immunogenic function to elicit NAb responses similar to HCMV. We also found that NAb raised against the PC by MVA-PC potently inhibit infection of Hofbauer cells, placental macrophages that are thought to be involved in virus circulation in utero.
 
By isolating monoclonal antibodies from MVA-PC vaccinated mice, we confirmed that the vector elicits NAb with antigen recognition and cell-type specific neutralization potency similar to human NAb recognizing conformational and linear epitopes of the PC and gH.  Furthermore, MVA-PC-derived NAb targeting the PC demonstrated potent ability to interfere with HCMV cell-to-cell spread in EC and infection of primary placental cytotrophoblasts, which are considered as key cell-type for HCMV transmission to the fetus. These results further validate MVA-PC as a vaccine candidate to induce NAb that potently inhibit HCMV host cell entry, cell-associated replication, and infection of cell types that are critical for HCMV vertical transmission.
 
Current work involves (1) development of an optimized MVA vaccine vector based on the PC and other dominant immunogens such as HCMV pp65, IE or gB to stimulate robust humoral and cell-mediated immune responses; (2) evaluation of NAb targeting the PC, gH/gL or a third gH/gL complex composed of gH/gL/gO and their mechanism to block HCMV entry into EC and FB; (3) functional and immunological characterization of neutralizing epitopes of the PC, gH/gL, or gH/gL/gO towards epitope-focused vaccine design; (4) expression of the gH/gL complexes in heterologous systems as a tool to study their structure and (5) refinement of HCMV vaccine design using the rhesus CMV/RM model to identify immune correlates of protection.

Publications

 
1.    Bialas KM, Tanaka T, Tran D, Varner V, Cisneros E, Chiuppesi F, Wussow F, Kattenhorn L, Macri S, Kunz EL, Estroff JA, Kirchherr J, Yue Y, Fan Q, Lauck M, O’Connor DH, Hall AHS, Xavier A, Diamond DJ, Barry PA, Kaur A, Permar S.  Maternal CD4+ T cells protect against severe congenital CMV disease in a novel nonhuman primate model of placental CMV transmission. P Natl Acad Sci, 112(44):13645-50, 2015. PMC4640765
 
2.    Chiuppesi F, Wussow F, Johnson E, Bian C, Zhuo M, Rajakumar A, Barry PA, Britt WJ, Chakraborty R, Diamond DJ. Vaccine-Derived Neutralizing Antibodies to the Human Cytomegalovirus gH/gL Pentamer Complex Neutralize Infection of Primary Cytotrophoblasts. J Virol. 89(23):1184-11898, 2015. PMC4645301
 
3.    Swanson ED, Gillis P, Hernandez-Alvarado N, Fernández-Alarcón C, Schmit M, Zabeli JC, Wussow F, Diamond DJ, and Schleiss MR. Comparison of Monovalent Glycoprotein B with Bivalent gB/pp65 (GP83) Vaccine for Congenital Cytomegalovirus Infection in a Guinea Pig Model: Inclusion of GP83 Reduces Antibody Response but Provides Equivalent Protection Against Mortality. Vaccine. 33(32):4013-8, 2015. PMC4772145
 
4.    Gibson L, Barysauskas CM, McManus M, Dooley S, Lilleri D, Fisher D, Srivastava T, Diamond DJ, Luzuriaga K. Reduced frequencies of polyfunctional CMV-specific T cell responses in infants with congenital CMV infection. Journal of Clinical Immunology,  35(3):289-301, 2015. PMCID:4366322
 
5.    Wussow F, Chiuppesi F, Martinez J, Campo J, Johnson E, Flechsig C, Newell M, Tran E, Ortiz J, La Rosa C, Herrmann A, Longmate J, Chakraborty R, Barry PA, Diamond DJ. Human Cytomegalovirus Vaccine Based on the Envelope gH/gL Pentamer Complex. PlosPathogens, 10(11):e1004524, 2014. PM: 25412505
 
6.    Gillis PA, Hernandez-Alvarado N, Gnanandarajah JS, Wussow F, Diamond DJ, Schleiss MR. Development of a novel, guinea pig-specific IFN-gamma ELISPOT assay and characterization of guinea pig CMV GP83-specific cellular immune responses following immunization with an MVA-vectored GP83 vaccine. Vaccine, 32(31):3963-3970, 2014.  PM24856783
 
7.    Gil A, Shen S, Coley S, Gibson L, Diamond DJ, Wang S, Lu S. DNA vaccine prime followed by boost with live attenuated virus significantly improves antigen-specific T cell responses against human CMV. Hum.Vaccin.Immunother. 9(10):2120-2132, 2013.  PM:24051429
 
8.    Wussow F, Yue Y, Martinez J, Deere JD, Herrmann, A., Longmate, J., Barry PA, Diamond DJ. A Vaccine Based on the Rhesus Cytomegalovirus UL128 Complex Induces Broadly Neutralizing Antibodies in Rhesus Macaques. J.Virol., 87(3):1322-1332, 2013.  PMC3554163
 
9.    Assaf B, Mansfield K, Westmoreland S, Kaur A, Oxford K, Diamond DJ, and Barry P. Patterns of Acute Rhesus Cytomegalovirus (RhCMV) Infection Predict Long-Term RhCMV Infection. J. Virol., 86: 6354-6357,2012. PMC3372188
 
10.    Oxford KL, Strelow L, Yue Y, Chang WL, Schmidt KA, Diamond DJ, Barry PA. Open reading frames carried on UL/b' are implicated in shedding and horizontal transmission of rhesus cytomegalovirus in rhesus monkeys. J Virol. (10):5105-14, 2011. PMC3126184
 
11.    Abel K, Martinez J, Yue Y, Lacey SF, Wang Z, Strelow L., Dasgupta A, Li Z, Schmidt  KA, Oxford KL, Assaf B, Longmate JA, Diamond DJ, and Barry PA. Vaccine-Induced Control of Viral Shedding following Rhesus Cytomegalovirus Challenge in Rhesus Macaques. J. Virology,  85(6):2878-2890, 2011. PMC3067943
 
12.    Yue Y, Wang Z, Abel K, Li J, Strelow L, Mandarino A, Eberhardt MK, Schmidt KA, Diamond DJ, Barry PA. Evaluation of recombinant modified vaccinia Ankara virus-based rhesus CMV vaccines in rhesus macaques. MMI, 197(2):117-123, 2008. PM:18196272
 
13.    Wang Z, La Rosa C, Maas R, Ly H, Brewer J, Mekhoubad S, Daftarian P, Longmate J, Britt WJ, and Diamond DJ. Recombinant MVA expressing a soluble form of Glycoprotein B causes durable immunity and neutralizing antibodies against multiple strains of CMV. J. of Virology, 78(8): 3965-3976, 2004. PMC374285
 
Project members:  Felix Wussow, Ph.D., Flavia Chiuppesi, Ph.D., Sona Chowdhury Ph.D., Joy Martinez, Zhuo Meng, Jenny Nguyen, Rana Chakraborty, M.D. and Erica Johnson, Ph.D., collaborators at Emory University Medical Center, and Don J. Diamond, Ph.D.
 
 
 

Funding

 
This work has been supported by funds from the National Cancer Institute (R29CA070819 and R21CA114889), a contract from SAIC (25X5061), FAMRI, Champion Power Equipment, the City of Hope Phase I program, and the City of Hope Cancer Center (P30-CA033572).

Personnel

 
Dr. Nicola Hardwick, Mary Carrol R.N., Mario DiMacali, C.R.A., Dr. Teodora Kaltcheva, Dr. Dajun Qian, Dr. Dean Lim, Dr. Lucille Leong, Dr. Peiguo Chu, Dr. Joseph Kim, Dr. Joseph Chao, Dr. Marwan Fakih, Dr. Yun Yen, Dr. Jonathan Espenschied, Dr. Joshua D I Ellenhorn, Dr. Don J. Diamond and Dr. Vincent Chung
 

Opportunities

 
For inquiries about this project or employment opportunities, please contact Melanie Lampa.
 
 

References

 
Hardwick N, Carrol M, Kaltcheva T, Qian D, Lim D, Leong L, Chu P, Kim J, Chao J, Fakih M, Yen Y, Espenschied J, Ellenhorn DI, Diamond DJ, Chung V.  2014.  p53MVA therapy in patients with refractory gastrointestinal malignancies elevates p53-specific CD8+ T cell responses. Clinical Cancer Research, 20(17):4459-4470.
    
Hardwick N, Chung V, Cristea M, Ellenhorn JDI, Diamond DJ.  2014.  Overcoming Immuno-suppression to enhance a p53MVA vaccine: Ain’t no mountain high enough. OncoImmunology.
    
Song GY, Srivastava T, Ishizaki H, Lacey SF, Diamond DJ, Ellenhorn JD. 2011. Recombinant modified vaccinia virus ankara (MVA) expressing wild-type human p53 induces specific antitumor CTL expansion. Cancer Invest. 29(8):501-10.
    
Ishizaki H, Song GY, Srivastava T, Carroll KD, Shahabi V, Manuel ER, Diamond DJ, Ellenhorn JD. 2010. Heterologous prime/boost immunization with p53-based vaccines combined with toll-like receptor stimulation enhances tumor regression. Journal of Immunotherapy 33(6):609-17
    
Song GY, Gibson G, Haq W, Huang EC, Srivasta T, Hollstein M, Daftarian P, Wang Z, Diamond D, Ellenhorn JD. 2007. An MVA vaccine overcomes tolerance to human p53 in mice and humans. Cancer Immunol Immunotherapy 56(8):1193-205.
    
Daftarian P, Song GY, Ali S, Faynsod M, Longmate J, Diamond DJ, Ellenhorn JD. Two distinct pathways of immuno-modulation improve potency of p53 immunization in rejecting established tumors. 2004. Cancer Res. 64(15):5407-14.
    
Espenschied J, Lamont J, Longmate J, Pendas S, Wang Z, Diamond DJ, Ellenhorn JD. CTLA-4 blockade enhances the therapeutic effect of an attenuated poxvirus vaccine targeting p53 in an established murine tumor model. 2003. J Immunol. 170(6):3401-7.