16 August 2023 Episode 343 Human Cytomegalovirus trends in management and vaccination

Episode 343: Towards a vaccine for human cytomegalovirus (HCMV)

 

Dear colleagues,

My attention was captured by a preprint on a promising mRNA vaccine for HCMV (see Ep 343-13 ).  To frame these early results well, one needs to understand the importance of HCMV and the challenges to control this nasty virus.

See

Par 1 Immuno-virology 

(Ep 343-1 Crough Clin Microbiol Rev 2009 ; Ep 343-2 Gerna Vaccines MDPI 2019; Ep 343-3 Gabrielle Stack Bitesized Immunology)

 

Cellular tropism = very broad

• Hematogenous cells: CD34+ progenitors (source of latent virus), myeloid cells (monocytes/macrophages and immature dendritic cells), lymphocytes.
• Other somatic cells found to be infected in disseminated infections: endothelial cells, epithelial cells, human fibroblasts, and smooth muscle cells.

Viral entry = complex:

1. Trimeric complex (TC) of glycoproteins H and L with O (gH/gL/gO or UL75/UL115/UL74)) for infection of all cell types.
2. Pentameric complex (PC) gH/gL with UL128, UL130, and UL131 for leuko- and endothelial tropism

 

Involving (at least) two cellular receptors:

• PDGFR-α (platelet-derived growth factor alpha) on fibroblasts, with additional receptors Transforming Growth Factor Receptor beta III (TGFRbIII) and neuregulin-2 (NRG2)
• Nrp2 (Neuropilin2) on endothelial/epithelial cells with additional receptors CD147, CD46, and OR14I1

 

gB is the fusion protein

 

Models of HCMV entry into human cells using the trimer complex (TC) or pentameric PC complexes.

 

 

 

TC interacts with PDGFRα via gO, then gH/gL/gO activates the pH-independent fusogenic activity of gB at the plasma membrane of fibroblasts.

PC first binds Nrp2, then the gH/gL component of the PC (or gH/gL/gO) induces endocytosis of virus particles], which are released into cytoplasm from endosomal membranes, following low-pH-induced activation of the gB fusion machinery

 

Life cycle

 

HCMV enters human cells either through direct fusion or through the endocytic pathway to release nucleocapsids into the cytoplasm.

These nucleocapsids are translocated into the nucleus, where viral DNA is released. This initiates the expression of IE-1/IE-2 genes. Viral replication and maturation follow the stimulation and parallel accumulation of viral synthesis function. This process involves the encapsulation of replicated viral DNA as capsids, which are then transported from the nucleus to the cytoplasm.

 Secondary envelopment occurs in the cytoplasm at the endoplasmic reticulum (ER)-Golgi intermediate compartment. This is followed by a complex two-stage final envelopment and egress process that leads to virion release by exocytosis at the plasma membrane.

 

Role of immediate early (IE), early (E) and late (L) genes

 

 

 

HCMV enters the cell through interaction of the host receptors with specific viral glycoproteins. Capsid and tegument proteins are release into the host cytosol. The capsid releases the viral genome into the nucleus, leading to the expression of immediate early (IE) genes.

• The IE proteins activate the expression of the early (E) genes.
• The E proteins initiate viral genome replication and the expression of late (L) genes.
• The L gene expression initiates the capsid assembly and the expression of tegument-and glycoproteins.

The genome loaded capsid enters the cytosol via nuclear egress. The capsid associates with the tegument proteins. The capsid acquires the viral envelope by budding into intracellular vesicles. The enveloped viral particles are released into the extracellular space.

 

 

Immune control and evasion of HCMV by innate and adaptive immunity.

 

The adaptive immune response contributes to the long-term control of HCMV.

 

 

 

• An extremely large cytotoxic HCMV-spec CD8 T cell response (up to 30 % of all CD8 T cells) is crucial for the control of primary HCMV infection and reactivation from latency.
• HCMV-spec IFN-γ-secreting CD4 T cells decrease virus shedding into urine, thus limiting its spread to new hosts.
• B cells also play a  role in controlling HCMV infection and may also help prevent its transmission to new hosts as maternal antibodies can confer protection to fetuses in utero.

 

More in detail:

 

Primary infection with HCMV in healthy individuals typically initiates with replication in mucosal epithelium (A), after which the virus disseminates to monocytic cells of myeloid lineage including monocytes and CD34 (+)cells, where it establishes latent infection (B). Restricted viral gene expression is observed in these latently infected cells, thus limiting their immune recognition by effector cells.

The differentiation of these virus-infected monocytes into macrophages can initiate productive infection (C). Virus particles or virus-associated dense bodies can be processed by professional antigen-presenting cells (e.g., DCs), which can stimulate antigen-specific T cells (D).

In addition, these DCs activated through Toll-like Receptors TLRs can also secrete a range of  cytokines and chemokines, which activate the innate arm of the immune system (e.g., NK cells) (D).

Virus-infected macrophages can also directly stimulate antigen-specific T cells (C). These activated T cells

(CD8+, CD4+, and/or γδ T cells) and NK cells can directly lyse virus-infected cells by cytolysis or block virus replication through the secretion of cytokines such as IFN-γ and/or TNF (E).

Another important arm of adaptive immunity involves B cells, which are also activated by the professional

antigen-presenting cells and control extracellular virus through antibody-mediated neutralization (F).

Distribution of CD4+ and CD8+ T-cell responses within HCMV-encoded proteins.

 

 

 

(A) Relative strengths of T-cell responses directed toward HCMV-encoded proteins with respect to expression kinetics (left) or gene function (right).

(B) Schematic representation of the magnitude of CD4+ and CD8+ T-cell responses against immunodominant HCMV-encoded proteins.

 

However: HCMV is a master of immune evasion

 

 

 

Nevertheless, despite the plethora of immune evasion mechanisms, there is a robust immune response to this virus. This is clearly demonstrated by the fact that most primary infections in immunocompetent host are asymptomatic and that HCMV disease occurs mainly in those with compromised or immature (e.g; foetal) immune systems.

 

Correlates of protection (CoP) ? (Ep 343-4 Cody Nelson JID 2020)

 

Based on animal models, natural history, experimental vaccines, no single CoP can be defined, but there are arguments for a role of

• Antibodies against free virus: both IgA (anti-acquisition) and IgG with neutralizing and non-neutralizing functions.  The non-neutralizing Ab are supposed to activate complement, Natural Killer (NK) cells and/or phagocytic cells.
• Cellular immunity: CD4+ and CD8+ T cells as well as NK cells: active against  cell-associated virus

 

According to the various stages of the disease and goals of the intervention

 

 

 

 

Par 2 Epidemiology: HCMV is very common (Ep 343-5 Zuhair Rev Med Virol. 2019)

 

 

The global prevalence of HCMV is very high, with regional differences from 66 % in Europe to 90 % in Eastern Mediterranean

 

Country specific mean seroprevalence and 95% uncertainty interval in women of reproductive age

(meta-regression–based estimates)

 

 

 

Clearly, several countries in Western Europe  score below 60 %, while many countries in Asia (including China and Turkey) and Africa score above 90 %.  

 

Congenital CMV (cCMV): (Ep 343-6 Karen Fowler Seminars Neonatology 2018)

Occurs in about 5–7 per 1000 live births overall. 

• Mother has a primary infection:
• Super-infection is responsible in about 50 % of cases: seropositive mothers exposed to individuals excreting HCMV (e.g. other babies in the family with primary infection ) or reactivation.

 

 

 

Par 3 Clinical aspects of HCMV infection itself  (Ep 434-7 Griffiths Antivir Res 2020 and Ep 343-8 Mayo Clinic 2023)

 

Primary infection: first infection in an individual with no immunity → the virus establishes latency

Reactivation from latency .

Reinfection = superinfection of someone who has already been infected, despite their natural immunity.

 

Any of these three subtypes of infection

• can complicate pregnancy, making HCMV the commonest cause of congenital infection.
• can produce severe disease in patients who are profoundly immunocompromised

 

 

 

 

 

Symptoms:

 

• Congenital (infection in utero): in 10-15 % symptomatic
  • Prematurity, low birth weight
  • Hepato-spleno-megaly, jaundice, liver insufficiency
  • Pneumonia
  • Petechia and/or rash
  • Microcephaly and seizures.
  • Hearing loss, poor vision, delayed development

 

• “Healthy” adults: usually no or mild flu-like: fatigue, fever, sore throat, muscle aches

 

• Immune compromised subjects: untreated HIV, immune suppression after solid organ transplant (SOT) or hematopoietic Stem Cell Transplant (SCT)
  • Pneumonia
  • Encephalitis
  • Retinitis
  • Hepatitis
  • Enteritis

 

 

 

 

 

Difference in risk between renal and bone marrow:

1. Renal transplant

• Major risk donor is seropositive and recipient seronegative (D + R-) → 70% of patients acquire primary infection
• Low-risk: donor is seronegative and recipient seropositive (D-R+) → 40% of patients reactivate latent virus
• Intermediate: both donor and recipient are seropositive (D + R+)  → 54% risk of viraemia, due to either reactivation of recipient virus or reinfection from the donor.

2. Hematopoietic stem cell transplant: risk is reversed

• Major risk comes from seropositive recipients reactivating CMV post-transplant.
• A seropositive donor makes a minor contribution to transmitting virus to recipients: In fact, there is evidence that seropositive donors may adoptively transfer some natural immunity into the recipient

 

Immune suppressive therapy increases viral load, while corticosteroid increase susceptibility of organs to HCMV infection.

 

 

Par 4 Role of HCMV in various inflammatory diseases, cancers  and in immune ageing

 

Role in inflammatory diseases and cancers  (Ep 343-9 Lele Ye Front Microbiol 2020)

 

Long term HCMV infection correlates with chronic inflammation in immunocompetent subjects = co-factor in the development of

• cardiovascular diseases
• auto-immune diseases
• some types of cancers

 

Primary and active infection causes serious diseases in immunotolerant individuals, such as arteriosclerosis, colitis,

esophagitis, colorectal cancer, gastric cancer, idiopathic thrombocytopenic purpura (ITP), prostate cancer,

systemic lupus erythematosus (SLE), systemic sclerosis (SSc), autoimmune connective tissue disease, neuroglioma, breast cancer, and retinitis

 

 

 

 

Model of age- and HCMV-related immune-senescence. (Ep 343-10 Wenjuan Tu Front Microbiol 2016)

 

 

Effect of age: Thymic involution contributes to reduced naïve Tcell export and Tcell diversity with age. To compensate existing naïve T cells increase homeostatic turnover.

Upon antigen stimulation, a large number of terminally differentiated CD45RA+CD8+ TEM cells clonally expand in the elderly, which dominate the memory pool and further restrict repertoire diversity.

These senescent CD45RA+ memory T cells typically have diminished  Tcell responses to stimulation in the absence of CD28 co-stimulatory signaling pathways and are characterized by a variety of altered transcriptional profiles which are epigenetically regulated (including by DNA methylation, histone modifications, micro RNAs, and chromatin remodeling).

 

In addition, HCMV infection can result in CD4+ naïve pool depletion and memory inflation, which further accelerate immunosenescence in aged individuals. In HCMV-infected elderly individuals, the CD8+ T cell response to HCMV antigens occupies nearly 50% of the entire memoryCD8+ T cell compartment in peripheral blood, while approximately 30% of total circulating CD4+ T cells can be HCMV-responsive

 

Together, age and HCMV infection  contribute to the overall decline in immune function, which might contribute to increased susceptibility to infectious disease and impaired immune responses to vaccination in the elderly.

 

 

Par 5 Treatment (Ep 343-11 Shiu-Jau Chen Trop Med Infect Dis 2022)

 

1. Drug treatment for acute infection (e.g. in transplant patients)

 

 

 

Novel drugs with less problems of side-effects and less cross-resistance

 

 

 

2. Immunotherapy

 

 

 

 

T cell therapy: MHC-peptide tetramer enrichment of HCMV-specific T cells or in vitro stimulation of T cells with HCMV viral lysate, recombinant viral vectors, synthetic peptides or mRNA, encoding HCMV antigens. Following enrichment or in vitro expansion, these cells are adoptively transferred into immunocompromised individuals either as a prophylactic or therapeutic treatment.

 

Alternatively, patients-derived dendritic cells, in vitro pulsed with HCMV antigens, could be adoptively transferred in  vivo

 

Par 6 Vaccine development (Ep 343-12 Xintao Hu Annu Rev Virol 2022)

 

1. Live attenuated vaccine:

• The HCMV strains AD169, Towne and Toledo: Ab induction lower than natural infection. No safety issues discovered, but theoretical risk on latent infection.
• Replication-defective HCMV vaccines, such as the V160 vaccine based on the AD169 strain: induced humoral and cellular responses similar to natural infection.  In phase 2 vaccine efficacy reached only 42.4% in the three-dose group and 32.0% in the two-dose group

 

Obvious candidates for recombinant technology:

• Three complexes involved in entry:

 

 

• The major tegument protein pp65, a major target for T cell immunity

 

 

 

 

• Immediate Early (IE) are also sometimes included: they are a target for T cells and by definition expressed early in the cycle → cells where reactivation occurs could be recognized and eliminated early on. 

 

Antibody induction against surface binding and fusion complexes → important for prophylactic vaccination in CMV seronegatives by either classical neutralization or non-neutralizing (complement and Fc receptor mediated activities 

 

T cell induction against structural epitopes → important for therapeutic vaccination in already infected (seropostives) subjects to prevent reactivation from latency

 

Antibody-dependent, non-neutralizing functions that may have contributed to gB/MF59 vaccine efficacy

 

 

A, Complement-dependent cytotoxicity (CDC). Antibodies bind to viral proteins on the surface of infected cell, then are cross-linked by c1q. This action causes an enzymatic cascade, culminating in the assembly of the membrane attack complex in the infected cell membrane.

B, Antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent respiratory burst (ADRB), results from antibody binding a viral protein on an infected cell, then engaging the Fc receptor on an immune effector cell. This immunologic bridge triggers release of cytotoxic granules, which destroy the infected cell.

C, Antibody-dependent cellular phagocytosis (ADCP) occurs when an antibody binds a virion then engages the Fc receptor on a phagocyte, triggering engulfment (and presumed destruction) of the virion.

 

 Abbreviations: DCs, dendritic cells; NK, natural killer.

 

 

2. Recombinant protein: gB (fusion protein) has been used in combination with adjuvant MF59 in phase 2  → Efficacy  against HCMV acquisition in seronegatives was 50% in postpartum women and 45 % in  adolescent females (→ potential strategy to prevent congenital CMV).

 

3. DNA vaccine:  VCL-CT02/ ASP0113, encoding ectodomain of gB and major tegumen protein pp65: elicited memory T cell response but minimal humoral response in HCMV-seronegative vaccinees in phase 1. Now tested in hematopoietic stem cell transplant patients.

 

4. Virus-like particles: group antigen (Gag) gene of murine leukemia virus and the plasmid DNA expressing fusion between HCMV gB and the G envelope protein of Vesiculo Stomatitis Virus (VSV) → induced high neut titers against gB in seronegative subjects (phase 1)

 

5. Viral vectors: primarily inducing T cell immunity against pp65, alone or combined with other CMV proteins

• Pox: ALVAC-CMV = canarypox with CMV pp65 and HCMC-MVA = Modified Ankara with pp65, IE1-exon 4, and IE2-exon5 (IE = immediate early genes)
• AVX601 vaccine is a bivalent alphavirus replicon vaccine expressing HCMV gB and pp65/IE1 fusion protein
• HB-101 vaccine is composed of two replication-deficient lymphocytic choriomeningitis viruses expressingHCMVpp65 and gB

 

6. mRNA by Moderna: mRNA-1647 vaccine composed of one mRNA from gB and five mRNAs from Pentameric complex and the mRNA-1443, composed of pp65.  

 

Ep 343-13: Xintao Hu medRxiv 8 August 2023:  mRNA-1647 Vaccine Candidate Elicits

• Potent and Broad  Neutralization
• and Higher Antibody-Dependent Cellular Cytotoxicity Responses  
• than that of the Partially Effective gB/MF59 Vaccine
• in both seronegative and seropositive subjects.

 

mRNA-1647 vaccination

• in seropositives boost of pre-existing HCMV-specific IgG responses, including neutralizing and Fc-mediated effector antibody responses (ADCC and ADCP).
• In seronegative vaccinees, induced durable and functional HCMV-specific IgG  responses: neutralization, ADCC and ADCP.
• Elicited gB-specific IgG responses were lower than the Pantamer Complex (PC)-specific IgG responses.

 

Long-lasting antigen-specific IgG antibody responses in seronegatives

 

 

Limited effect in seropositives

 

Durable neutralizing antibody responses by mRNA-1647 in both seropositive and seronegative vaccine recipient

 

ID50 = 50 % neutralization titers against Towne HCMV in fibroblasts and against AD169r HCMV in fibroblasts and epithelial cells

 

Most pronounced effect in seronegatives, but also clear increase of neut Ab in seropositives

 

Efficient induction of both antibody-dependent cytotoxicity (ADCC) and phagocytosis (ADCP)  in seronegatives

 

 

 

Levels in seronegatives lower than in seropositives

 

 

Comparison of gB-specific peak IgG binding antibody and neutralizing antibody responses.

 

 

 

 

The recombinant protein gB8 + MF59 adjuvant ad compared to mRNA-1647 induces:

• Higher levels of gB binding IgG
• Lower levels of neutralizing Ab
• Rather similar levels of ACCC and ADCP

 

 

General conclusions

 

HCMV is a chronic infection that may seem innocent. However:

1. It can cause serious generalized clinical problems in newborns (congenital) and immunocompromised subjects.
2. In immunocompetent subjects it may contribute to a variety of cardiovascular, inflammatory and neoplastic conditions.  In addition it is an important factor in immune ageing, with increased susceptibility to infections and decreased response to any vaccination.
3. Drug treatment (Ganciclovir, Cidofovir, Foscarnet)  used to be limited by side effects and cross-resistance, but novel promising drugs (Letermovir, Maribavir) are becoming available.
4. There is some progress in vaccination,

• for prophylactic use Ab-induction against fusion protein gB and the pentameric entry complex
• for therapeutic vaccines T cell epitopes