MULTIVALENT CMV VACCINE AND USES THEREOF
20210260184 · 2021-08-26
Inventors
Cpc classification
A61K39/295
HUMAN NECESSITIES
C12N2710/16134
CHEMISTRY; METALLURGY
C12N2710/16122
CHEMISTRY; METALLURGY
International classification
Abstract
The invention is directed to multivalent HCMV immunogenic compositions and their use.
Claims
1. A multivalent composition comprising at least two antigenic human cytomegalovirus (hCMV) polypeptides or nucleic acids or a portion thereof, wherein the antigens or a portion thereof are of at least two different genotypes.
2. A composition comprising hCMV gB5 polypeptide or nucleic acid sequence (of
3. A multivalent composition comprising at least two hCMV gB polypeptides or nucleic acids encoding gB antigens or a portion thereof, wherein the gB antigens or a portion thereof are of at least two different genotypes, at least three different genotypes, at least four different genotypes, or at least five different genotypes, wherein the genotypes are gB1, gB2, gB3, gB4, or gB5.
4. The composition of claim 3 wherein the at least two different genotypes are: hCMV gB1 and hCMV gB2, hCMV gB1 and hCMV gB3, hCMV gB1 and hCMV gB4, hCMV gB1 and hCMV gB5, hCMV gB2 and hCMV gB3, hCMV gB2 and hCMV gB4, hCMV gB2 and hCMV gB5, hCMV gB3 and hCMV gB4, hCMV gB3 and hCMV gB5, hCMV gB4 and hCMV gB5, or any combination thereof.
5. The composition of claim 3 wherein the at least three different genotypes are: hCMV gB1, hCMV gB2 and hCMV gB3; hCMV gB1, hCMV gB2 and hCMV gB4; hCMV gB1, hCMV gB2 and hCMV gB5; hCMV gB2, hCMV gB3 and hCMV gB4; hCMV gB2, hCMV gB3 and hCMV gB5; hCMV gB3, hCMV gB4 and hCMV gB5, or any combination thereof.
6. The composition of claim 3 wherein the at least four different genotypes are: hCMV gB1, hCMV gB2, hCMV gB3 and hCMV gB4; hCMV gB1, hCMV gB2, hCMV gB3 and hCMV gB5; hCMV gB2, hCMV gB3, hCMV gB4 and hCMV gB5; hCMV gB1, hCMV gB3, hCMV gB4 and hCMV gB5, or any combination thereof.
7. The composition of claim 3 wherein the at least five different genotypes are hCMV gB1, hCMV gB2, hCMV gB3, hCMV gB4 and hCMV gB5.
8. The composition of any one of claim 3-7 wherein the nucleic acid is mRNA.
9. A composition comprising at least one nucleic acid encoding at least one hCMV antigen of at least two different genotypes, at least three different genotypes, at least four different genotypes, or at least five different genotypes, wherein the antigen is gB and the genotypes are gB1, gB2, gB2, gB3, gB4, or gB5, and wherein the nucleic acid is formulated in at least one lipid nanoparticle (LNP).
10. A vector comprising a nucleic acid encoding any one of the antigens of the invention.
11. A host cell comprising a nucleic acid encoding any one of the antigens of the invention.
12. A cell culture comprising any of the host cells of the invention.
13. A method of inducing an immune response against hCMV comprising administering to a subject in need thereof a composition of the invention.
14. Methods of inducing immune responses using the compositions of the invention.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0043] To conform to the requirements for PCT applications, many of the figures presented herein are black and white representations of images originally created in color.
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DETAILED DESCRIPTION
[0066] The genomes of over 20 different HCMV strains have been sequenced, including those of both laboratory strains and clinical isolates. For example, the following strains of HCMV have been sequenced: Towne (GL239909366), AD169 (GI:219879600), Toledo (GL290564358) and Merlin (GI: 155573956). HCMV strains AD169, Towne and Merlin can be obtained from the American Type Culture Collection (ATCC VR538, ATCC VR977 and ATCC VR1590, respectively).
[0067] Cytomegalovirus contains a number of membrane protein complexes. Of the approximately 30 known glycoproteins in the viral envelope, gH and gL have emerged as particularly interesting due to their presence in several different complexes: dimeric gH/gL, trimeric gH/gL/gO (also known as the gCIII complex), and the pentameric gH/gL/pUL128/pUL130/pUL131 (pUL131 is also referred to as “pUL131A”, “pUL131a”, or “UL131A”; pUL128, pUL130, and pUL131 subunits sometimes are also referred as UL128, UL130, UL131). CMV is thought to use the pentameric complexes to enter epithelial and endothelial cells by endocytosis and low-pH-dependent fusion but it is thought to enter fibroblasts by direct fusion at the plasma membrane in a process involving gH/gL or possibly gH/g/gO. The gH/gL and/or gH/gL/gO complex(es) is/are sufficient for fibroblast infection, whereas the pentameric complex is required to infect endothelial and epithelial cells.
[0068] The pentameric complex is considered as a major target for CMV vaccination. See US Pub 20180028645A1. Viral genes UL128, UL130 and UL131 are needed for endothelial entry (Hahn, Journal of Virology 2004; 78:10023-33). Fibroblast-adapted non-endothelial tropic strains contain mutations in at least one of these three genes. Towne strain, for example, contains a two base pair insertion causing a frame shift in UL130 gene, whereas AD169 contains a one base pair insertion in UL131 gene. Both Towne and AD169 could be adapted for growth in endothelial cells, and in both instances, the frame shift mutations in UL130 or UL131 genes were repaired.
[0069] U.S. Pat. No. 7,704,510 discloses that pUL131A is required for epithelial cell tropism. U.S. Pat. No. 7,704,510 also discloses that pUL128 and pUL130 form a complex with gH/gL, which is incorporated into virions. This complex is required to infect endothelial and epithelial cells but not fibroblasts. Anti-CD46 antibodies were found to inhibit HCMV infection of epithelial cells.
[0070] CMV vaccines tested in clinical trials include Towne vaccine. Towne-Toledo chimeras, an alpha virus replicon with gB as the antigen, gB/MF59 vaccine (see Pass, R F, et al., N Engl J Med 2009; 360:1191-9), a gB vaccine produced by GlaxoSmithKline, and a DNA vaccine using gB and pp65 (See Tang et al. Hum Vaccin Immunother. 2017 Dec. 2; 13(12):2763-2771. doi: 10.1080/21645515.2017.1308988. Epub 2017 May 11). pp65 is viral protein that is a potent inducer of CD8+ responses directed against CMV. These vaccines are all poor inducers of antibodies that block viral entry into endothelial/epithelial cells (Adler, S. P. (2013), British Medical Bulletin, 107, 57-68. doi:10.1093/bmb/Idt023).
[0071] Preclinical animal studies in CMV vaccines include an inactivated AD169 which has been repaired in the UL131 gene, a DNA vaccine using a wild-type UL130 gene and peptide vaccines using peptides from pUL130 and 131 (Sauer, A, et al., Vaccine 2011; 29:2705-1, doi:10.1016).
[0072] A recombinant Human Cytomegalovirus (HCMV) gL protein vaccine candidate is provided in US Pub 20170369532.
[0073] Glycoprotein B (gB) is a homotrimer protein, 907 amino acids in length. It is relatively conserved among herpesviruses and is essential for entry into all cells. Many gB-specific antibodies are neutralizing, though the majority are non-neutralizing. See Burke H G, Heldwein E E (2015) Crystal Structure of the Human Cytomegalovirus Glycoprotein B. PLoS Pathog 11(10): e1005227.
[0074] However, there still remains a need for a safe and effective CMV vaccine.
[0075] Genetic variability of CMV is well known, with well documented different strains, genotypes and subtypes. See Murthy et al. (2011) Detection of a Single Identical Cytomegalovirus (CMV) Strain in Recently Seroconverted Young Women. PLoS ONE 6(1): e15949. doi.org/10.1371/journal.pone.0015949. Numerous studies have examined a potential link between CMV virulence and pathogenesis, particularly in the context of solid organ transplantation, and sequence polymorphism of the viral genome. See e.g. Pang et al. Am J Transplant. 2009 February; 9(2):258-68. doi: 10.1111/j.1600-6143.2008.02513.x., without finding of clear correlation between sequence polymorphism and viral pathogenesis, see Sarcinella et al. J Clin Virol. 2002 February; 24(1-2):99-105, Humar et al. J Infect Dis. 2003 Aug. 15; 188(4):581-4. Epub 2003 July 29.
[0076] As disclosed and exemplified herein, analyzing gB/MIF59 vaccines and placebo recipient samples (see Example 1), the inventors have discovered an enrichment of gB1 genotype HCMV variants among placebo recipients (7/13 placebo recipients vs. 0/5 gB vaccines), which suggests that the gB1 genotype vaccine antigen may have elicited genotype-specific protection. These data indicate that gB immunization may have had a measurable impact on viral intrahost population dynamics. These finding suggest that including multiple HCMV genotypes in a multivalent vaccine composition may achieve breadth of HCVM protection.
[0077] CMV genetic diversity is known and characterized. For a discussion of CMV genotypes see e.g. Murthy et al. (2011) Detection of a Single Identical Cytomegalovirus (CMV) Strain in Recently Seroconverted Young Women. PLoS ONE 6(1): c15949. doi.org/10.1371/journal.pone.0015949: Pang et al. Am J Transplant. 2009 February; 9(2):258-68. doi: 10.1111/j.1600-6143.2008.02513.x., specifically reference to Chou and Dennison at second paragraph in the introduction: Coaguette et al. Clin Infect Dis. 2004 Jul. 15; 39(2):155-61. Epub 2004 Jun. 23, specifically at discussion and references in second paragraph in introduction, all of which content is incorporated by reference in its entirety.
[0078] In some embodiments, the antigens are administered as recombinant proteins. In other embodiments the antigens are administered as nucleic acids. Non-limiting embodiments include mRNA, including but not limited to modified mRNA. See e.g. US Pub 20180028645A1.
[0079] Genetic variability of CMV antigens can be determined by those of ordinary skill in the art by obtaining sequence data and aligning the amino acid and/or nucleic sequences using readily available and well-known alignment algorithms (such as BLAST, using default settings; ClustalW2; or algorithm disclosed by Corpet, Nucleic Acids Research, 1998, 16(22):10881-10890).
[0080] Pharmaceutical Compositions and Administration
[0081] In certain aspects the invention provides pharmaceutical compositions comprising the CMV proteins and nucleic acids of the invention.
[0082] The diverse CMV proteins and nucleic acids described herein can be incorporated into an immunogenic composition, or a vaccine composition. Such compositions can be used to raise antibodies in a mammal (e.g. a human).
[0083] The invention provides pharmaceutical compositions comprising the CMV proteins and nucleic acids described herein, and processes for making a pharmaceutical composition involving combining the CMV proteins and nucleic acids described herein with a pharmaceutically acceptable carrier. The pharmaceutical compositions of the invention typically include a pharmaceutically acceptable carrier, and a thorough discussion of such carriers is available in Remington: The Science and Practice of Pharmacy.
[0084] The pH of the composition is suitable for physiological use, and is usually between about 4.5 to about 11. Stable pH may be maintained by the use of a buffer e.g. a Tris buffer, a citrate buffer, a phosphate buffer, or a histidine buffer. Thus a composition will generally include a buffer.
[0085] A composition may be sterile and/or pyrogen free. Compositions may be isotonic with respect to humans.
[0086] A composition comprises an immunologically effective amount of its antigen(s). A skilled artisan can readily determine the effective amount.
[0087] Immunogenic compositions may include an immunological adjuvant. Exemplary adjuvants include mineral-containing compositions; oil emulsions; saponin formulations; virosomes and virus-like particles; bacterial or microbial derivatives; bioadhesives and mucoadhesives; liposomes; polyoxyethylene ether and polyoxyethylene ester formulations; polyphosphazene (pcpp); muramyl peptides; imidazoquinolone compounds; thiosemicarbazone compounds; tryptanthrin compounds; human immunomodulators; lipopeptides; benzonaphthyridines; microparticles: immunostimulatory polynucleotide (such as RNA or DNA: e.g., CpG-containing oligonucleotides).
[0088] For example, the composition may include an aluminum salt adjuvant, an oil in water emulsion (e.g. an oil-in-water emulsion comprising squalene, such as MF59 or AS03), a TLR7 agonist (such as imidazoquinoline or imiquimod), or a combination thereof. Suitable aluminum salts include hydroxides (e.g. oxyhydroxides), phosphates (e.g. hydroxyphosphates, orthophosphates), (e.g. see chapters 8 & 9 of Vaccine Design (1995) eds. Powell & Newman. ISBN: 030644867X. Plenum), or mixtures thereof. The salts can take any suitable form (e.g. gel, crystalline, amorphous, etc.), with adsorption of antigen to the salt being an example.
[0089] One suitable immunological adjuvant comprises a compound of Formula (I) as defined in WO2011/027222, or a pharmaceutically acceptable salt thereof, adsorbed to an aluminum salt. Many further adjuvants can be used, including any of those disclosed in Powell & Newman (1995).
[0090] Compositions may include an antimicrobial, particularly when packaged in multiple dose format. Antimicrobials such as thimerosal and 2 phenoxyethanol are commonly found in vaccines, but sometimes it may be desirable to use either a mercury-free preservative or no preservative at all.
[0091] Compositions may comprise detergent e.g. a polysorbate, such as polysorbate 80. Detergents are generally present at low levels e.g. <0.01%.
[0092] Compositions may include sodium salts (e.g. sodium chloride) to give tonicity. A concentration of 10+−2 mg/ml NaCl is typical, e.g., about 9 mg/ml.
[0093] In certain embodiments, where the composition comprises nucleic acids such as mRNA, whether modified or unmodified, the mRNAs could be formulated in lipid nanoparticles (LNPs). See US Pub 20180028645A1 and WO 2015/164674), the content of which is incorporated by reference in its entirety.
[0094] In another aspect, the invention provides a method of inducing an immune response against cytomegalovirus (CMV), comprising administering to a subject in need thereof an immunologically effective amount of the immunogenic composition, which comprises the proteins, DNA molecules, RNA molecules (e.g., self-replicating RNA molecules, mRNAs), or virus-like replicon particles (VRPs) as described herein. In certain embodiments the compositions are administered to provide protection against congenital CMV infection. In certain embodiments the compositions are administered to provide protection against CMV infection in immunocompromised individuals, transplant recipients, or during organ transplantation.
[0095] In certain embodiments, the immune response comprises the production of neutralizing antibodies against CMV. In certain embodiments, the neutralizing antibodies are complement-independent.
[0096] The immune response can comprise a humoral immune response, a cell-mediated immune response, or both. In some embodiments an immune response is induced against each delivered CMV protein. A cell-mediated immune response can comprise a Helper T-cell (Th) response, a CD8+ cytotoxic T-cell (CTL) response, or both. In some embodiments the immune response comprises a humoral immune response, and the antibodies are neutralizing antibodies. Neutralizing antibodies block viral infection of cells. CMV infects many cell types, including epithelial cells and also fibroblast cells. In some embodiments the immune response reduces or prevents infection of both cell types. Neutralizing antibody responses can be complement-dependent or complement-independent. In some embodiments the neutralizing antibody response is complement-independent. In some embodiments the neutralizing antibody response is cross-neutralizing; i.e., an antibody generated against an administered composition neutralizes a CMV virus of a strain other than the strain used in the composition.
[0097] Compositions of the invention will generally be administered directly to a subject. Direct delivery may be accomplished by parenteral injection (e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly, or to the interstitial space of a tissue), or by any other suitable route. For example, intramuscular administration may be used e.g. to the thigh or the upper arm. Injection may be via a needle (e.g. a hypodermic needle), but needle-free injection may alternatively be used. A typical intramuscular dosage volume is about 0.5 ml.
[0098] Dosage can be by a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunization schedule and/or in a booster immunization schedule. In a multiple dose schedule the various doses may be given by the same or different routes, e.g., a parenteral prime and mucosal boost, a mucosal prime and parenteral boost, etc. Multiple doses will typically be administered at least 1 week apart (e.g., about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, etc.).
[0099] The subject may be an animal, preferably a vertebrate, more preferably a mammal. Exemplary subject includes, e.g., a human, a cow, a pig, a chicken, a cat or a dog, as the pathogens covered herein may be problematic across a wide range of species. Where the vaccine is for prophylactic use, the human is preferably a child (e.g., a toddler or infant), a teenager, or an adult; where the vaccine is for therapeutic use, the human is preferably a teenager or an adult. A vaccine intended for children may also be administered to adults, e.g., to assess safety, dosage, immunogenicity, etc.
[0100] Vaccines of the invention may be prophylactic (i.e. to prevent disease) or therapeutic (i.e. to reduce or eliminate the symptoms of a disease). The term prophylactic may be considered as reducing the severity of or preventing the onset of a particular condition. For the avoidance of doubt, the term prophylactic vaccine may also refer to vaccines that ameliorate the effects of a future infection, for example by reducing the severity or duration of such an infection.
[0101] Isolated and/or purified CMV proteins, complexes, and nucleic acids described herein can be administered alone or as either prime or boost in mixed-modality regimes, such as a RNA prime followed by a protein boost. Benefits of the RNA prime protein boost strategy, as compared to a protein prime protein boost strategy, include, for example, increased antibody titers, a more balanced IgG1:IgG2a subtype profile, induction of TH1-type CD4+ T cell-mediated immune response that was similar to that of viral particles, and reduced production of non-neutralizing antibodies. The RNA prime can increase the immunogenicity of compositions regardless of whether they contain or do not contain an adjuvant.
[0102] In the RNA prime-protein boost strategy, the RNA and the protein are directed to the same target antigen. Examples of suitable modes of delivering RNAs include virus-like replicon particles (VRPs), alphavirus RNA, replicons encapsulated in lipid nanoparticles (LNPs) or formulated RNAs, such as replicons formulated with cationic nanoemulsions (CNEs). Suitable cationic oil-in-water nanoemulsions are disclosed in WO2012/006380 e.g. comprising an oil core (e.g. comprising squalene) and a cationic lipid (e.g. DOTAP, DMTAP, DSTAP, DC-cholesterol, etc.).
[0103] An RNA prime-protein boost regimen may involve first (e.g. at weeks 0-8) performing one or more priming immunization(s) with RNA (which could be delivered as VRPs, LNPs, CNEs, etc.) that encodes one or more of the protein components of a CMV protein complex of the invention and then perform one or more boosting immunization(s) later (e.g. at weeks 24-58) with: an isolated CMV protein complex of the invention, optionally formulated with an adjuvant or a purified CMV protein complex of the invention, optionally formulated with an adjuvant. In some embodiments, the prime and boost vaccine comprise the same immunogen(s). In some embodiments, the prime and boost vaccine comprise difference immunogens.
[0104] In some embodiments, the RNA molecule is encapsulated in, bound to or adsorbed on a cationic lipid, a liposome, a cochleate, a virosome, an immune-stimulating complex, a microparticle, a microsphere, a nanosphere, a unilamellar vesicle, a multilamellar vesicle, an oil-in-water emulsion, a water-in-oil emulsion, an emulsome, a polycationic peptide, a cationic nanoemulsion, or combinations thereof.
[0105] Also provided herein are kits for administration of nucleic acid (e.g., RNA), purified proteins, and purified nucleic acids described herein, and instructions for use. The invention also provides a delivery device pre-filled with a composition or a vaccine disclosed herein.
[0106] The pharmaceutical compositions described herein can be administered in combination with one or more additional therapeutic agents. The additional therapeutic agents may include, but are not limited to antibiotics or antibacterial agents, antiemetic agents, antifungal agents, anti-inflammatory agents, antiviral agents, immunomodulatory agents, cytokines, antidepressants, hormones, alkylating agents, antimetabolites, antitumor antibiotics, antimitotic agents, topoisomerase inhibitors, cytostatic agents, anti-invasion agents, antiangiogenic agents, inhibitors of growth factor function inhibitors of viral replication, viral enzyme inhibitors, anticancer agents, .alpha.-interferons, .beta.-interferons, ribavirin, hormones, and other toll-like receptor modulators, immunoglobulins (Igs), and antibodies modulating Ig function (such as anti-IgE (omalizumab)).
[0107] In certain embodiments, the compositions disclosed herein may be used as a medicament, e.g., for use in inducing or enhancing an immune response in a subject in need thereof, such as a mammal.
[0108] In certain embodiments, the compositions disclosed herein may be used in the manufacture of a medicament for inducing or enhancing an immune response in a subject in need thereof, such as a mammal.
[0109] One way of checking the efficacy of therapeutic treatment involves monitoring pathogen infection after administration of the compositions or vaccines disclosed herein. Another way of checking the efficacy of prophylactic treatment involves monitoring immune responses, systemically (such as monitoring the level of IgG1 and IgG2a production) and/or mucosally (such as monitoring the level of IgA production), against the antigen. Typically, antigen-specific serum antibody responses are determined post-immunization but pre-challenge whereas antigen-specific mucosal antibody responses are determined post-immunization and post-challenge.
[0110] This invention is further illustrated by the following examples which should not be construed as limiting.
EXAMPLES
Example 1
[0111] Example 1: Genetic signatures of human cytomegalovirus variants acquired by seronegative glycoprotein B vaccines
[0112] Human cytomegalovirus (HCMV) is the most common congenital infection worldwide, and a frequent cause of hearing loss or debilitating neurologic disease in newborn infants. Thus, a vaccine to prevent HCMV acquisition is a public health priority. The glycoprotein B (gB)+MF59 adjuvant subunit vaccine is the most efficacious tested clinically to date, demonstrating approximately 50% efficacy in multiple phase 2 trials. Yet, the impact of gB/MF59-elicited immune responses on the population of viruses acquired by trial participants has not been assessed. In this analysis, we employed quantitative PCR as well as two distinct next-generation sequencing strategies (short amplicon and whole gene) to interrogate genetic differences between the HCMV populations infecting gB/MF59 vaccines and placebo recipients. For the majority of subject-specific viral populations analyzed, we identified a single dominant genetic haplotype along with a large number of minor haplotypes present at low frequency. This finding suggests that the intrahost viral population constitutes a heterogeneous swarm of genetically-distinct viral variants. Additionally, we identified several possible distinctions between the viral populations of acutely-infected vaccines and placebo recipients. First, there was reduced magnitude peak viral shedding in the saliva of gB vaccines compared to placebo (median copies/mL: placebo=319, gB/MF59=51; p=0.02, Exact Wilcoxon Rank Sum test). Furthermore, employing a panel of tests for genetic compartmentalization, we noted evidence of tissue-specific gB haplotypes in 3 of 4 vaccines, though only in 1 of 7 placebo recipients. Finally, we observed an enrichment of gB1 genotype HCMV variants among placebo recipients (7/13 placebo recipients vs. 0/5 gB vaccines), which suggests that the gB1 genotype vaccine antigen may have elicited genotype-specific protection. These data indicate that gB immunization may have had a measurable impact on viral intrahost population dynamics and support future analysis of a larger cohort.
[0113] Human cytomegalovirus (HCMV) congenital infection affects 1 in 150 pregnancies (1) and is the most frequent non-genetic cause of sensorineural hearing loss and neurodevelopmental delay in infants worldwide (2). Additionally, HCMV is the most common infectious agent among allograft recipients, often causing end-organ disease such as hepatitis, pneumonitis, or gastroenteritis and predisposing the individual to graft rejection (3). It has been estimated that an efficacious HCMV vaccine would save the United States 4 billion dollars and 70,000 quality-adjusted life years annually, and thus HCMV vaccine development has remained a tier 1 priority of the National Academy of Medicine for the past 17 years (4).
[0114] The glycoprotein B (gB)+MF59 adjuvant vaccine is the most efficacious HCMV vaccine platform trialed to date, demonstrating partial vaccine protection in multiple patient populations. In a cohort of HCMV-seronegative postpartum women, gB vaccination achieved a promising 50% vaccine efficacy (5). When this study was subsequently repeated in a cohort of seronegative adolescent women, a comparable level of vaccine-protection was observed (6). Furthermore, in allograft recipients, the same gB vaccine reduced duration of HCMV viremia and antiviral therapy (7). The mechanism of this partial vaccine protection remains unknown, though we and others have observed this vaccine platform was particularly poor at eliciting heterologous neutralizing antibodies in these populations and that non-neutralizing antibody responses may have played a role. See Nelson et al. Proc Natl Acad Sci USA. 2018 Apr. 30. pii: 201800177. Yet any distinction between viruses acquired by gB/MF59 vaccines and placebo recipients has not been thoroughly evaluated, and this remains a critical question for understanding the functional antiviral immunity responsible for the partial vaccine efficacy demonstrated in these clinical trials.
[0115] With a genome consisting of 236 kilobase pairs (8) and encoding approximately 164 open reading frames (9), HCMV has the largest genome of any human virus. Thus, prior to the advent of whole-genome sequencing, it was extraordinarily challenging to assess HCMV viral population composition and diversity because of the limitations of traditional sequencing methodologies. Nevertheless, it is now well established that HCMV is highly polymorphic between and within individuals, defined via a variety of sequencing methodologies including restriction fragment length polymorphism analysis (10), targeted gene sequencing (11-16), and whole genome sequencing (17-19). Yet the source of this diversity remains poorly understood. If multiple unique viral variants are identified in a single individual (so-called “mixed infection”), does this represent de novo mutations, simultaneous initial infection with multiple unique variants, or independent, sequential infection events?Mixed infections have been frequently detected in both chronically HCMV-infected individuals (20) and immunocompromised hosts (12-14). Yet, recently-seroconverted women from the gB/MF59 vaccine trial predominantly had a single variant detected in all tissues and at all time points (16) when evaluated by a traditional Sanger sequencing methodology, suggesting that mixed infections in healthy individuals may result from independent, sequential infection events.
[0116] One major limitation of traditional HCMV genotyping to quantify HCMV diversity is a lack of sensitivity to detect viral variants present at low frequency. Recent HCMV whole-genome, next-generation sequencing (NGS) has suggested that there is remarkable intrahost diversity, comparable to many RNA viruses, stemming from the presence of low-frequency alleles representing minor viral variants (17-19). Thus, it has been established that HCMV likely exists within individual hosts as a heterogeneous population of unique, but related viral variants (19). Subsequent characterization of the intrahost composition and distribution of low-frequency viral variants, has led to recognition of unique viral populations between individual organs representing anatomic compartmentalization of viral populations (18).
[0117] As HCMV diversity is due to the presence of distinct, low-frequency viral variants, traditional sequencing methodologies may not be the most appropriate means to discern differences between intrahost viral populations. Thus, we applied a previously-validated (21) sequencing methodology and analysis pipeline termed Short NGS Amplicon Population Profiling (SNAPP) to investigate the viral populations of recently-seroconverted gB/MF59 vaccines and placebo recipients. This technique, which employs sequencing of an approximately 500 base-pair region at tremendous read depth, has facilitated a more complete understanding of in vivo viral dynamics. We hypothesize that gB/MF59 vaccination limited the complexity of the in vivo viral population following primary HCMV infection. Metrics of HCMV population dynamics including viral load, pairwise genetic diversity, number of unique haplotypes (viral variants), and the characteristics of those variants can be studied in concert with vaccine-elicited immune responses to arrive at a more comprehensive understanding of the mechanism of partial vaccine efficacy.
[0118] Results:
[0119] Viral Load, Number of Haplotypes, and Haplotype Sequence Diversity by Vaccine Group
[0120] We obtained HCMV DNA extracted from plasma, saliva, urine, and vaginal fluid of 53 gB/MF59 vaccine and placebo recipients following HCMV primary infection. Samples were taken approximately monthly, though sampling was heterogeneous between trial participants. Peak plasma viral load (
[0121] Next, short (˜550 base pair), variable regions within gB and UL130 (membrane glycoprotein targeted by neutralizing antibodies, but not included in gB/MF59 vaccine) were amplified by nested PCR then deep-sequenced (
[0122] The peak nucleotide diversity (π) for each patient was calculated for identified haplotypes at the gB (
[0123] Viral Load, Number of HCMV Haplotypes, and Sequence Diversity by Anatomic Compartment.
[0124] For compartment analysis, all samples (including from chronically HCMV-infected seropositive individuals) were combined. The peak HCMV viral load for each patient in each anatomic compartment was compared (
[0125] Presence and Persistence of Low-Frequency, Unique HCMV Variants.
[0126] The relative frequency of unique viral haplotypes was identified for tissue culture virus stocks, chronically HCMV-infected individuals, and primary HCMV-infected gB/MF59 vaccines and placebo recipients at the gB (
[0127] Anatomic Compartmentalization of HCMV Populations in gB/MF59 Vaccines.
[0128] A panel of tests for genetic compartmentalization reliant upon 6 distinct distance and tree-based methods was employed to assess the extent to which viral populations in different anatomic compartments of a single subject constitute distinct populations. Given our definition of compartmentalization based on significant results for at least 3 of 6 tests, anatomic compartmentalization at the gB locus was observed for 1 of 7 placebo recipients, 3 of 4 gB vaccine recipients, and 0 of 4 chronically HCMV-infected individuals (
[0129] gB Genotype Analysis.
[0130] In addition to gB and UL130 SNAPP to define viral haplotypes, the full gB ORF was amplified, fragmented, and sequenced by NGS to identify a gB consensus sequence for each unique sample (
[0131] Additionally, we inferred a phylogenetic tree using sequences from the full gB ORF (
TABLE-US-00001 TABLE 1 Distinct gB genotypes detected in various clinical samples from placebo recipients and gB/MF59 vaccines using different sequencing methodologies. gB cleavage Full gB ORF gB Patient Group site Sanger NGS SNAPP 1 Placebo 4 4, 5 1, 4, 5 2 Placebo 3 3 1, 3 3 Placebo 3 3 1, 3 4 Placebo 1 1 1, 5 5 Placebo 2 2 1, 2, 5 6 Placebo 1 — 1 7 Placebo 2 — 2 8 Placebo 1 1 1 9 Placebo 1 1 — 10 Placebo 1 1 — 11 Placebo 1, 4 1, 4 1, 4 12 Placebo 1 1 1 14 Placebo 1 1 1 15 Placebo 5 5 1, 5 17 Placebo 5 5 5 22 gB/MF59 3 3 1, 3, 5 25 gB/MF59 1 5* 1 27 gB/MF59 3 3.sup.+ 1, 3 30 gB/MF59 5 5 1, 5 32 gB/MF59 5 5 1, 5
DISCUSSION
[0132] Despite the partial efficacy demonstrated by gB/NIF59 vaccination in multiple clinical trials (5-7), there has been little examination of the impact of this vaccine on the in vivo viral populations. In this investigation, we sought to employ the enhanced sensitivity of next-generation sequencing (NGS) technology to delve deeper into the question of whether there are discernable differences between viruses acquired by gB/MF59 vaccines and placebo recipients. The advantage of NGS over more traditional sequencing methodologies is the ability to detect minor viral variants, which contribute to the diversity of the overall viral population (
[0133] Throughout the study, we identified several indications of vaccine-mediated effects on the viral population. First, peak HCMV shedding in saliva was reduced by an order of magnitude in gB/MF59 vaccines, suggesting that the vaccine responses may limit viral replication in salivary glands. gB/MF59 vaccination is known to elicit high titers of gB-specific IgG, IgA, and SIgA in parotid saliva (24), which may have suppressed HCMV salivary replication and reduced saliva viral shedding. However, there was no difference observed in peak systemic viral load or peak viral load in urine and vaginal fluid between infected vaccines and placebo recipients. Since women were only tested for HCMV acquisition every 3 months and since the time-point of infection and viral load/viral shedding kinetics are unknown, it is possible that sampling limitations may have obscured any differences between groups.
[0134] Secondly, we observed that 3 of 4 gB vaccines with viral DNA available from multiple compartments exhibited evidence of viral genetic compartmentalization at the gB locus, in contrast to only 1 of 7 placebo recipients. As has been previously described (16), we observed that the dominant viral variant was identical between anatomic compartments in the majority of subjects. The evaluation of gB-specific compartmentalization was therefore only discernable because of the ability of Short NGS Amplicon Population Profiling (SNAPP) to detect minor viral variants. Our data are consistent with HCMV whole-genome NGS indicating tissue-specific variants, with intrahost variable SNPs at relatively low frequency (17, 18). The mechanism leading to the observed compartmentalization in vaccines is unclear, though it is possible this phenomenon stems from either neutral or positive selection in distinct anatomic compartments. One hypothesis is that systemic gB-specific antibodies prevented unrestricted dissemination of HCMV variants between tissue compartments. Then, this bottleneck might have reduced founder population size and increased the speed and likelihood of stochastic fixation of neutral SNPs and formation of genetically-distinct viral populations (25, 26). Alternatively, it is possible that local factors including cell type and local antibody production/secretion at the site of HCMV replication selected for “more fit” viral variants in each compartment.
[0135] Finally, we observed an interesting trend that among viruses for which the full gB ORF was sequenced, 7 of 13 placebo recipients (54%) and 0 of 5 gB/MF59 vaccines (0%) acquired a gB1 genotype virus (p=0.08, Fishers Exact test). If we instead consider the acquisition of genetically-similar gB genotypes gB1, gB2, and gB4 between vaccines and placebo recipients, there is statistically-significant inhibition of acquisition of these genotypically-homologous viruses (gB/MF59=10/13, Placebo=0/5; p=0.03, Fisher's Exact test). Of note, the gB immunogen in this vaccine trial was based on the Towne strain (gB1 genotype prototypic sequence) suggesting the possibility of vaccine genotype-specific protection. Complementarily, we have observed in the same gB/MF59 vaccine cohort that gB-elicited neutralization activity was only detectable against the autologous Towne strain virus, but not heterologous viruses AD169 and TB40/E (Nelson et al. Proc Natl Acad Sci U S A. 2018 Apr. 30. pii: 201800177. doi: 10.1073/pnas.1800177115. [Epub ahead of print] PubMed PMID: 29712861). These observations raise the possibility of gB1 genotype-specific protection against HCMV acquisition based on neutralization of only the autologous virus. Furthermore, as demonstrated by force-of-infection modeling, gB1 genotype-specific protection could explain the 50% partial vaccine efficacy observed in this phase 2 clinical trial. The concept of neutralization breadth has not been explored extensively for HCMV, though several papers have described strain-specific neutralization (27-29). Of note, low frequency gB1 genotype haplotypes were detectable in several vaccines by SNAPP, suggesting there may not have been a true barrier to gB1 genotype acquisition but rather restricted gB1 genotype virus replication.
[0136] By far the largest limitation of this study was sample availability. Unfortunately, the scope of our investigation was restricted by: 1) the original sampling timeline employed during the clinical trial, 2) the availability of clinical samples, and 3) the integrity of the DNA more than a decade following DNA extraction. Additionally, as with any study based on PCR amplification and DNA sequencing, there is a potential for primer bias, contamination, and background error to obscure the results. We instituted several measures to increase data integrity. First, primers were designed and validated to prevent amplification bias (21). Additionally, PCR, sequencing, and analysis was completed in duplicate to reduce the likelihood of contamination affecting results. An advantage of this investigation is that we were able to validate our two sequencing methodologies (SNAPP and full gB ORF NGS) by comparing observed gB genotypes with previously published data (16). The gB genotype predicted by Sanger sequencing and NGS sequencing methodologies were identical for 88% of all samples. However, because of the relatively small cohort size and potential for sequencing error, our observed trends certainly merit further investigation.
[0137] Nonetheless, this investigation is the first to employ NGS of viral DNA from infected gB/MF59 vaccine and placebo recipients in an attempt to characterize the viral determinants of HCMV acquisition. Our observation of reduced saliva shedding and a high rate of gB sequence compartmentalization in vaccines suggests an impact of gB-elicited antibodies on viral population dynamics. Furthermore, the observation of possible vaccine immunogen genotype (gB1)-specific protection is intriguing, and, when paired with our previous finding that gB/MF59 vaccination elicited neutralization of the autologous (gB1) Towne strain virus but not heterologous virus strains in this same trial (Nelson et al. Proc Natl Acad Sci USA. 2018 Apr. 30. pii: 201800177. doi: 10.1073/pnas.1800177115. PubMed PMID: 29712861), strongly implies that strain-specificity of the immune response may have played a role in vaccine protection. Thus, the impact of including multiple gB genotypes in next-generation HCMV vaccines should be investigated in subsequent studies.
[0138] Materials and Methods:
[0139] Study population. The study population was comprised of 53 postpartum women who acquired HCMV infection while participating in a phase 2, randomized, double-blind, placebo controlled clinical trial of an HCMV vaccine (16). Clinical trial participants were HCMV-seronegative, healthy postpartum women immunized with gB protein (based on Towne, gB-1 genotype) vaccine (Sanofi Pasteur) with MF59 adjuvant (Novartis) on a 0, 1 and 6 month schedule and were screened for HCMV infection every three months for 3.5 years using an antibody assay which detects seroconversion to CMV proteins other than gB (30). Institutional review board (IRB) approval was obtained from University of Alabama at Birmingham and Johns Hopkins Hospital and all subjects signed an approved consent form. The Duke University Health System determined that analysis of de-identified samples from these cohorts does not constitute human subjects research.
[0140] Viral isolation. Subjects with serologic evidence of infection were tested for HCMV in blood, urine, saliva and vaginal swab from one month to 3.5 years after seroconversion. Aliquots of each specimen were stored at −80° C. Fresh urine, saliva and vaginal swab specimens were inoculated into cultures of MRC-5 cells (ATCC) or locally prepared human foreskin fibroblasts. Cultures were checked weekly for 4-6 weeks after inoculation; CMV was identified by its characteristic cytopathic effect. Tissue culture with CMV (primary isolate or first passage) was frozen at −80° C. for later analysis.
[0141] DNA extraction and Quantitative PCR. Total genomic DNA was extracted from infected cells using a capture-column kit (Qiagen, Valencia, Calif.). HCMV DNA was extracted from 400 μL of original samples—blood, urine, saliva, or vaginal swab in culture media—using the MagAttract virus mini M48 kit (Qiagen) on Biorobot M48. The quantitative PCR assay is based on amplification of a 151-bp region from the US17 gene (23, 31). As previously reported, the limit of detection is 100 copies/mL (4 copies/well), with a measurable range of 100 to 10.sup.8 copies per mL.
[0142] Short NGS amplicon population profiling (SNAPP). Flow chart detailing the sequencing strategy is shown in
TABLE-US-00002 TABLE 2 Primer sequences. Primer Sequence fullgB_PCR1_F 5′-ACACGCAAGAGACCACGACG-3′ fullgB_PCR1_R 5-TTGAAAAACATAGCGGACCG-3′ fullgB_PCR2_F 5′-ATGGAATCCAGGATCTGGTG-3′ fullgB_PCR2_R 5′-TCAGACGTTCTCTTCTTCGT-3′ gBamp_PCR2_F 5′-Illumina_overhang-GAAAA CAAAACCATGCAATT-3′ gBamp_PCR2_R 5′-Illumina_overhang-GTCGG ACATGTTCACTICTT-3′ UL130_PCR1_F 5′-TGGGATGGGTGCAGAAGGT-3′ UL130_PCR1_R 5′-GGCTTCTGCTTCGTCACCAC-3′ UL130_PCR2_F 5′-Illumina_overhang-ATCGC ACCTGAAAAGACACG-3′ UL130_PCR2_R 5′-Illumina_overhang-CCCCG CCATGGTCTAAACTG-3′
[0143] Full glycoprotein B open reading frame PCR and sequencing. Flow chart denoting sequencing strategy is shown in
[0144] SNAPP haplotype reconstruction and nucleotide diversity. Data processing flow chart is shown in
[0145] Where L=sequence length in nucleotides for π. N=Total number of reads in sample, dij=Number of nucleotide differences between haplotype i and j, fi=Number of reads belonging to haplotype i, π.sub.NS and rrs were calculated as the average dS and dN between pairs of haplotypes weighted by the haplotypes abundance:
[0146] Where L=sequence length in amino acids for πN, πS, N=Total number of reads in sample, dSij=dS between haplotype i and j sequences, fi=Number of reads belonging to haplotype i. Correlations were performed between various measures of viral population diversity (viral load, number of haplotypes, π, π.sub.S, and πi.sub.N), and suggest that haplotypes, π, π.sub.S, and πi.sub.N are somewhat related measures although are not directly equivalent (
[0147] Phylogenetic trees and genotype assignment. Protein or nucleotide sequences of interest were aligned using the ClustalW algorithm (40) in Mega (version 6.06) (41). A neighbor-joining tree was constructed using the Los Alamos National Labs “neighbor treemaker” (accessed at Los Alamos Database hiv.lanl.gov/components/sequence/HIV/treemaker/treemaker.html), then the tree was plotted in FigTree (version 1.4.3). Full gB ORF sequences were assigned to gB genotypes based on phylogenetic proximity to reference gB sequences from GenBank. Because of sample limitation, if the full gB assigned genotype did not match the genotype assigned to SNAPP amplicons and/or previously published Sanger sequencing data (16), these sequences were omitted from the phylogenetic analysis.
[0148] Force-of-infection modeling. The cohort of women in the postpartum gB/MF59 vaccine trial were predominantly African-American (>70%) (5), and thus we utilized an HCMV force-of-infection estimate for non-Hispanic, African-American individuals of 5.7 per 100 persons (42). Additionally, we made the assumption that gB1 genotype viruses comprise 54% of the circulating virus pool, based on 6 of 11 placebo recipients acquiring gB1 genotype viruses. Finally, we hypothesized that gB1-vaccinated individuals were universally protected against all circulating gB1 genotype viruses. Modeling was done using Matlab, and source code is included in the supplementary material.
REFERENCES FOR EXAMPLE 1
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Example 2
[0191] Animal Studies
[0192]
[0193] Animal studies could assess the strength and breadth of immune responses, types of antibodies responses, protection or any other suitable determination that measures responses and/or efficacy.
[0194] Additional animal models, such as non-human primates or guinea pigs, could be used to assess whether different CMV genotypes achieve more breadth in the vaccine response, and/or improved responses such as neutralization, protection, ect.