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Studies of Adoptively Transferred CMV-Specific T Cells

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Method of Expansion/Selection

Riddell, 1992, 1995

Expansion using CMV-infected fibroblasts

Einsele, 2002

Expansion with CMV lysate

Cobbold, 2005

Tetramer Selection using magnetic beads

Micklethwaite, 2008

Antigen-presenting cells (Dendritic cells) transduced with an adenoviral vector encoding CMVpp65

Peggs, 2011

Selection of T cells secreting IFN-γ after exposure to CMV antigen

Blyth, 2013

Antigen-presenting cells (Dendritic cells) transduced with an adenoviral vector encoding CMVpp65 or Dendritic cells pulsed with HLA-A02-restricted peptide NLVPMVATV

Quoted from (Hanley and Bollard, 2014).

The ability to generate CMV, EBV, and adenovirus-specific CTL from the 20% fraction of a cord blood unit by using dendritic cells transduced with an Ad5/f35-CMV-pp65 vector as well as the cytokines IL-7, IL-12, and IL-15 was reported by Hanley and colleagues in 2009. Responding T cells were shown to be derived from the naïve T cell population and responded to typical and atypical, novel CMV-pp65 epitopes. Later on, the ability to generate CMV-specific T cells from CMV-seronegative donors was reported by Jedema et al., 2011 and Hanley et al., 2013.


On the basis of the cost to the health care system and the impact of the virus on human suffering, the development of an effective prophylactic vaccine to prevent CMV symptomatic congenital disease and/or to prevent disease in immunocompromised individuals is a high priority and would be a highly cost-effective measure (Khanna and Diamond, 2006). A successful vaccine strategy should aim to stimulate the innate and adaptive immune responses at the appropriate time. Both humoral and cell-mediated immune responses might be necessary to prevent congenital disease, whereas cellular immune response alone might be sufficient to prevent virus-associated complications in transplant patients (Khanna and Diamond, 2006).

Cytomegalovirus exhibits a high level of molecular diversity and carries many immune evasion genes (Hansen et al., 2010). Thus, infection within a host can occur with multiple virus strains concomitantly, including at the time of initial infection, or sequentially (Renzette et al., 2011). Broad and cross-neutralizing cellular and humoral responses have therefore become a major goal of vaccine design (Arvin et al., 2004).

Various strategies have been developed, though a vaccine against CMV remains elusive. CMV vaccines have been obtained using attenuated or chimeric viruses, DBs, recombinant proteins, DNA, peptides and/or viral vectors (poxvirus/adenovirus) (Khanna and Diamond, 2006). A number of subunit CMV vaccines tested in clinical trials targeted the abundant pp65 protein (Sylwester et al., 2005), which is expressed by CMV-infected cells both early and late after infection (La Rosa et al., 2012).

Cytomegalovirus vaccines in clinical trials include: glycoprotein B subunit vaccines; alphavirus replicon particle vaccines; DNA vaccines; and live-attenuated vaccines. A variety of vaccine strategies are also being examined in preclinical systems and animal models of infection. These include: recombinant vesicular stomatitis virus vaccines; recombinant modified vaccinia virus Ankara; replication-deficient adenovirus-vectored vaccines; and recombinant live-attenuated virus vaccines generated by mutagenesis of cloned rodent CMV genomes maintained as bacterial artificial chromosomes in Escherichia coli (Sung and Schleiss, 2010).

  1. Trial of a subunit vaccine consisting of recombinant HCMV envelope gB with MF59 adjuvant:

All HCMV-infected individuals have a significant proportion of neutralizing antibodies to HCMV being specific for epitopes on gB (Sung and Schleiss, 2010). A study of the use of HCMV gB vaccine plus MF59 adjuvant was reported. It was administered following a 0-, 1- and 6-month schedule (Pass et al., 2009).

Although the study demonstrated that the gB vaccine could significantly reduce the risk of acquiring primary maternal HCMV infection, the study did not address the question of whether vaccine-induced HCMV immunity was equivalent to natural immunity in modulating either infection rate or sequelae for the fetus (Dekker and Arvin, 2009).

Since re-infection with new strains of HCMV with which the host has no prior experience can lead to transmission to the fetus with subsequent sequelae (Boppana et al., 2001), the issue of cross-protection against diverse clinical isolates following administration of gB vaccine from a single genotype must be defined in future studies (Sung and Schleiss, 2010).

  1. Clinical trial evaluation of a two-component alphavirus replicon particle vaccine containing HCMV gB and phosphoprotein 65 (pp65)/immediate early fusion proteins:

The gB and the pp65 are the most frequently recognized antigens by CD4+ T cells, and pp65 is also one of the antigens most frequently recognized by CD8+ T cells (Sylwester et al., 2005). The HCMV IE1 is also an important target of the CD8+ T-cell response (Slezak et al., 2007). Therefore, vaccination strategies that aimed at eliciting T-cell responses has focused on the pp65 protein andIE1 gene product (Sung and Schleiss, 2010). AVX601 is a two-component alphavirus replicon particle vaccine expressing HCMV gB and a fusion protein of pp65-IE1 (Reap et al., 2007). The vaccine was well tolerated, with only mild local reactogenicity, Mild-to-moderate systemic reactogenicity was reported in some subjects (Sung and Schleiss, 2010).

  1. Bivalent HCMV DNA vaccine:

The use of a HCMV DNA vaccine in immunocompromised subjects, such as transplant recipients, would eliminate the safety concerns of live-attenuated HCMV or live recombinant viral-vectored vaccines (Selinsky et al., 2006). DNA vaccines elicit robust CD4+ and CD8+ T-cell and antibody responses (Sung and Schleiss, 2010). VCL-CB01, a bivalent HCMV DNA vaccine that contains two plasmids encoding HCMV pp65 and gB (Liu  and Ulmer, 2005).

This vaccine has the ability to prime antigen-specific T cells, with the capacity to proliferate and secrete IFN-γ on restimulation with antigen (Wloch et al., 2008). Further modifications of this vaccine may be required to optimize immunogenicity, particularly to the gB moiety (Sung and Schleiss, 2010). It was generally well tolerated. The most common adverse event was mild site injection pain (Liu and Ulmer, 2005).

  1. Live-attenuated HCMV Towne vaccine with or without adjuvant recombinant IL-12 and/or priming by DNA vaccine:

Immunization with Towne vaccine prevented HCMV disease in seronegative renal transplant recipients, although it did not prevent infection in these patients or in parents of HCMV-infected children (Sung and Schleiss, 2010).

Evidence suggests that the relative defect in Towne vaccine may be related to inadequate antigen-specific IFN-γ responses by CD4+ and CD8+ T cells following vaccination (Jacobson et al., 2006).

Approaches to improve the immunogenicity of the Towne vaccine are being explored (Jacobson et al., 2009). One approach was to generate genetic recombinant vaccines containing regions from the genome of the unattenuated Toledo strain of HCMV, substituted for the corresponding regions of the Towne genome (Heineman et al., 2006). In another approach, HCMV DNA vaccine is used to prime for memory immune responses to Towne vaccine (Jacobson et al., 2009). A third approach is to co-administer Towne with recombinant human IL-12 (Jacobson et al., 2006*).

5) Preclinical vaccine development

  1. Recombinant vesicular stomatitis virus expressing murine cytomegalovirus gB:

As a recombinant vaccine vector, vesicular stomatitis virus (VSV) can induce strong humoral and cellular immunity, particularly at mucosal surfaces. This attribute makes recombinant VSV (rVSV) an attractive candidate for development of a vectored HCMV vaccine (Wilson etal., 2008).

Live rVSV vector expressing a murine CMV homolog of the gB protein has been tested in the mouse model (Wilson etal., 2008). This induced neutralizing antibody responses, and resulted in reduced viral titers. Also, splenocytes from immunized mice produced a CD8+ IFN-γ response to gB (Sung and Schleiss, 2010).

  1. Recombinant modified vaccinia virus Ankara:

The attenuated poxvirus, modified vaccinia virus Ankara (MVA), was established as a safe and potent antigen delivery system. Its genome has undergone six major deletions during serial passage (Sung and Schleiss, 2010), which, in turn, allows the insertion of multiple HCMV genes (Wang et al., 2007).

A recombinant MVA vaccine that expresses a soluble, secreted form of HCMV gB, based on the AD169 strain sequence has been constructed (Wang et al., 2004). High levels of gB-specific neutralizing antibodies were elicited in vaccinated mice (Sung and Schleiss, 2010).

A trivalent MVA expressing gB, pp65 and IE1 has been developed (Wang et al., 2006) with ability to induce humoral and cellular immunity to gB (Wang et al., 2006). Recombinant MVAs have also been generated expressing both full-length pp65 and exon 4 of IE1 with induction of robust primary cell-mediated immunity and stimulation of vigorous expansion of memory Tcell responses to both antigens (Wang et al., 2007).

Another recombinant MVA expressing pp65 and a fusion protein of HCMV IE1 exon 4 and IE2 exon 5 was constructed to maximize the representation of IE-specific immunity (Wang et al., 2008).

Replication-deficient adenovirus-vectored polyepitope vaccine:

Systemic and mucosal immunity to MCMV could be induced by intranasal immunization using a replication deficient adenoviral vector expressing murine CMV glycoprotein H in a murine model (Shanley and Wu, 2005). Modified adenoviral vector Ad5F35, Ad5F35-AD-1, has been generated, expressing the immunodominant antigenic domain-1 epitope of HCMV gB based on the sequence from the AD169 strain (Zhao et al., 2009). Since the AD-1 epitope is well conserved between different strains of HCMV (Britt et al., 2005), expression of the AD-1 epitope from AD5F35 elicits neutralizing antibody responses to diverse clinical isolates (Zhao et al., 2009).

Another replication deficient adenoviral-vectored vaccine, Ad-gBCMVpoly (Zhong et al., 2008) which encodes 46 HCMV T-cell epitopes from multiple antigens covalently linked to the extracellular domain of HCMV gB antigen (Zhong et al., 2008). This chimeric vaccine elicited neutralizing antibody responses and virus-specific CD4+ and CD8+ T-cell responses (Zhong and Khanna, 2009).

  1. Recombinant live CMV vaccine by bacterial artificial chromosome mutagenesis:

An ideal live-attenuated HCMV vaccine should grow to high titers in cell culture for easy production, should be severely attenuated in vivo, even in immunocompromised hosts, and should elicit a strong immune response sufficient to protect against HCMV-associated disease (Mohr et al., 2008). An approach to the generation of such a vaccine is the targeted deletion of CMV genes modulating the host immune response (Cicin-Sain et al., 2007). This approach has been facilitated by the advances in mutagenesis of cloned CMV genomes maintained as bacterial artificial chromosomes in Escherichia coli as well as the rapidly expanding knowledge about the role of viral genes in immunopathogenesis and immune evasion (Dunn et al., 2003).

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