Insights Into Kaposi Sarcoma-Associated Herpesvirus-Specific Humoral Responses

Abstract

Kaposi sarcoma (KS) remains a global health concern. In sub-Saharan Africa, where there is a high burden of HIV-1 infection, there is also a high prevalence of infection by the etiologic agent of KS, the KS-associated herpesvirus (KSHV). Despite the successes of antiretroviral treatment (ART), the burden of KS and other KSHV-associated malignancies among people living with HIV under ART remained high, stressing the need for a greater understanding of the immune response against KSHV infection. Here, we review the current information on KSHV-specific humoral response in infected individuals in detail. We discuss the significance of anti-KSHV responses, mechanisms used by KSHV to subvert and modulate humoral immunity, and implications for pathogenesis and therapy. We highlight cutting-edge serological assays and bioinformatics tools that aid the development of effective vaccines against KSHV, underscoring the complexity of the humoral response and its critical role within the context of infection and vaccine design.

1 ∣. Introduction

Kaposi sarcoma (KS), as a multifocal pigmented sarcoma of the skin, was first described in 1872 by Dr. Moritz Kaposi [1, 2]. Despite being a rare form of cancer before the HIV epidemic, KS incidence increased significantly among HIV infected individuals who had developed AIDS before the introduction of antiretroviral therapy (ART). It is within the KS tissues from these AIDS patients that Drs. Yuan Chang and Patrick Moore discovered Kaposi sarcoma-associated herpesvirus (KSHV) in 1994, the eighth member of the human herpesvirus family (HHV-8), as the causative agent of KS [3]. Subsequent studies confirmed that KSHV was also the etiological agent of several other diseases, such as Primary Effusion Lymphoma (PEL), Multicentric Castleman Disease (MCD), and KSHV Inflammatory Cytokine Syndrome (KICS) [4-6].

The global prevalence of KSHV varies considerably, with sub-Saharan Africa (SSA) being one of the highest-impacted regions, where an estimated 20%–80% of the adult population is infected with KSHV [2, 7]. The risk of KSHV-associated diseases is also the highest in SSA, where the virus is endemic. Although KSHV infection occurs across different genders and age groups, specific populations such as people living with HIV (PLWH), men who have sex with men (MSM), the elderly, and organ transplant patients with compromised immune systems are at particular risk of developing KSHV-associated diseases. Despite adherence to ART, the incidence of KSHV-associated diseases, such as KS, is still high in SSA countries like Tanzania, Zambia, and Uganda [7]. Besides SSA, KSHV seroprevalence is also high among parts of the Mediterranean, South American, and specific Western Asian populations. Importantly, the US population is not immune to KSHV infection. The risks of developing KSHV-associated diseases, particularly KS, are the highest among HIV-infected individuals, especially in the southern states of the USA, where the HIV burden is high [8, 9]. Moreover, KS cases continue to arise among immunosuppressed solid organ transplant recipients, including instances of donor-derived KSHV transmission, underscoring the ongoing clinical relevance of KSHV within the US healthcare system.

KSHV can be transmitted through blood, sexual contact, or organ transplantation; however, the virus is spread primarily through saliva [10-12]. KSHV infection is known to have occurred early in life, as several studies reported that nearly 40% of children in SSA were infected by the age of 4 [13, 14]. Most of these infections happen via saliva contact, such as premastication, a common behavioral practice among mothers and caregivers in SSA. Mother-to-child transmission through breastfeeding is unlikely to be a route of early childhood infection in SSA [13].

Upon infection, KSHV quickly enters its latency stage, where only a few latency-related viral proteins, such as the latency-associated nuclear antigen (LANA), are expressed, allowing KSHV to evade the host immune surveillance [15, 16]. Most infected individuals may never develop any KSHV-related illnesses throughout their lives. Only after years of latent infection, in the event of a weakened immune surveillance, such as immunodeficiency by HIV infection, or organ transplantation, do KSHV-infected individuals eventually develop KS or other KSHV-related malignancies. Lessons from studies of other latency-prone viral diseases, such as HIV, highlighted the important role of the T-cell immune component in suppressing KS development. In fact, many groups in the KSHV field are currently focused on deciphering KSHV-specific T-cell responses in infected individuals with or without KS. On the other hand, although humoral response may not play a role in suppressing KS development since there are high levels of neutralizing antibodies (nAbs) in KS patients, humoral response is nonetheless an important but often neglected area of the KSHV field. As demonstrated by the recent vaccines against SARS-CoV-2, humoral response is critical in preventing infection. In the case of KSHV, potent vaccine-induced nAbs could, in theory, limit viral entry during primary exposure and thereby reduce the likelihood of widespread infection and latency establishment, as has been shown for certain other enveloped viruses such as hepatitis B virus (HBV) (against surface antigen HBsAg) and SARS-CoV-2 (against spike protein). This concept underlies the rationale for developing a prophylactic vaccine against KSHV. Unfortunately, there is no approved KSHV-specific vaccine to date. Hence, a better understanding of the humoral response elicited by KSHV may provide valuable insights into its role in preventing infection and pathogenesis, leading to the development of novel vaccines and therapeutic strategies. To this end, we present an up-to-date review on KSHV-specific humoral responses in infected individuals and the various methodologies utilized in KSHV serology research. We will also dive into the KSHV antigens that were shown to have elicited antibody (Ab) responses, the potential to target these antigens with a vaccine, and how advanced bioinformatics tools may help to improve vaccine designs. Together, this review will recap what the KSHV field has learned about KSHV-specific humoral responses and how this information can help us to prevent KSHV infection and its associated diseases.

2 ∣. Humoral Immune Response Upon Natural KSHV Infection

The detection of pathogen-specific antibodies (Abs) in serum or plasma (i.e. serology) has been the gold standard to determine whether an individual is exposed to the pathogen, including KSHV. KSHV-specific Abs may help limit viral dissemination or control reactivation within the host, but there is no evidence that naturally acquired antibodies generated after infection can prevent the establishment of latency. Moreover, the overall detectable humoral response to KSHV is inconsistent and highly variable [13, 17, 18]. This has complicated the monitoring of KSHV seroprevalence for numerous reasons, such as the occurrence of seroreversion [13, 18]. The cause of seroreversion is currently unknown, but one possibility is that periods of viral latency reduce detectable antigen levels, leading to diminished antibody titers. Importantly, this does not imply that continuous antigenic stimulation is required for durable humoral immunity, as shown in vaccinology, but rather that intermittent viral reactivation may shape the strength and detectability of KSHV-specific response. Consequently, there has been a push to better understand KSHV pathogenesis and the anti-KSHV humoral immune response, starting from its initial infection to the development of KSHV-related illnesses [19].

To understand the initial response to KSHV infection, most studies have focused on child cohorts in SSA, where KSHV is highly prevalent and KSHV-specific Abs can be detected early in life during initial infection. In Uganda, KSHV seroprevalence has been reported at 9% among 2-year-old children and 30.6% among 8-year-old children [20]. A Zambian study recorded 40% of children in the cohort seroconverting by 2 years of age [13]. Seroreversion was also considerably more common among children, with only 40% of KSHV seropositive Zambian children remaining seropositive over 24 months [13, 18]. Infant children of KSHV seropositive mothers have virus-specific antibodies within the first few months. These have been shown to originate from the mother through transplacental transfer of antibodies or through breastfeeding [21]. Children of KSHV seropositive mothers are shown to be protected from KSHV infection for as long as they are breastfed by their mother, suggesting that this transfer of antibodies offers temporary protection [22, 23]. However, breastfeeding offers similar levels of protection from KSHV seronegative mothers, suggesting that other protective factors from breastmilk may be protective, such as lactoferrin or complement proteins [22]. The study referenced the possibility that these seronegative mothers could have seroreverted during the study but transferred undetectable antibodies through breastmilk. Additional study on Zambian mother–children pairs found that only 1 of 86 children who remained seronegative with a seropositive mother had nAbs, indicating that any humoral protection from early infection is likely from non-nAb functions [23]. Nonetheless, these protective maternal Abs only persist for several months until the child weans from breastfeeding, and their susceptibility to KSHV infection increases thereafter. It is also possible that breastfeeding influences KSHV transmission risk through behavioral factors, for example, limiting alternative feeding practices such as premastication of food, which may increase exposure risk. A comprehensive longitudinal study is warranted to investigate both the immunological and behavioral components of mother-derived protection in infants.

Studies on the KSHV-specific Ab response in adults have provided numerous insights into KSHV and its associated diseases, such as the increase in the overall KSHV-specific Ab titer in KS patients compared to KSHV-infected asymptomatic individuals [17, 24, 25]. This disease-dependent increase is also seen in KSHV-specific nAbs levels, in which their presence is 66.7% prevalent among KS patients compared to only 6.5% in asymptomatic individuals [24]. The presence of nAbs in KS patients has also been found to be age-dependent, where older patients (> 30 years) are more likely to have nAbs present [24]. However, these differences in nAb levels have not been found to correlate with disease stage, treatment response, or HIV status [24, 26-28]. In addition to nAbs, non-nAbs may contribute to viral control through other mechanisms, such as binding to infected cells via their Fab regions, leaving their Fc regions exposed which is recognized by natural killer (NK) cells, macrophages, and other immune effector cells through their Fc receptors (FcγRs), leading to the destruction of the infected cells via various cell or complement-mediated effector functions. These include antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and antibody-dependent complement deposition (ADCD). Although KSHV-specific antibodies have been implicated in ADCC in-vitro, there is no clinical evidence to suggest that it plays any role in KS pathogenesis [29]. No studies have yet reported the ability of KSHV-specific Abs to elicit ADCP or ADCD. Given that ADCP was reported in Epstein–Barr Virus (EBV) and the structural and immunological similarities between EBV and KSHV, it is plausible that similar ADCP occurs after KSHV infection, which warrants further investigations [30].

Like other viruses, such as HIV, KSHV evolved the ability to escape host humoral responses through various mechanisms. Besides using glycosylation to shield its vital glycoproteins involved in cell entry from antibody recognition, KSHV has other unique mechanisms to further evade host humoral pressure after infection. One mechanism is altering the host's natural Ab class-switch recombination (CSR) process. CSR is a humoral mechanism that changes the effector function of an Ab while retaining its antigen specificity [31]. This involves B cells exchanging the IgM heavy chain constant (C) region for a downstream C-region that switches the isotype from IgM to another, such as IgG, IgE, or IgA. A mouse study revealed that KSHV vIL-6 protein functionally enhances immunoglobulin heavy chain CSR by increasing the expression of activation-induced cytidine deaminase [32]. Clinically, in HIV-associated (epidemic) KS, higher total (non-KSHV specific) serum IgA has been linked to progression to more advanced tumor stages [33, 34], consistent with HIV-driven polyclonal hypergammaglobulinemia rather than protective, antigen-specific immunity. In contrast, studies measuring KSHV-specific antibodies, particularly salivary IgA/IgG to latent and lytic antigens, found higher titers in patients with regressed disease [35], aligning with a role for mucosal antigen-specific responses in controlling a virus that reactivates and is shed orally. Thus, the prognostic meaning of “IgA” depends on what is measured (total vs. antigen-specific), where it is measured (serum vs. saliva), and clinical context (ART era, disease stage). In addition, IgE levels have been found to increase in KS patients compared to those without KS. This suggests that CSR to IgE leads to IgE-mediated atopic inflammation, which may contribute to KS pathogenesis [36]. While it is believed that there is an association between vIL-6 activity and these effects of Ig isotypes on KSHV pathogenesis, further studies are needed to fully understand the mechanisms involved and the role of HIV or HAART treatment on KSHV-related CSR.

Through working with KSHV-infected tonsillar B cells, Totonchy and colleagues discovered that KSHV can also cause a second occurrence of recombinase-activating gene-mediated recombination of the Ig light chain during B cell development outside of the standard bone marrow setting [37]. Usually, a second recombination of the Igλ locus only occurs if the first rearrangements of the Igκ locus fail to produce a functional, non-autoreactive B cell receptor, and this process is limited to the bone marrow. However, KSHV can induce rearrangement of the Igλ locus after infection in the circulation. This unique ability is hypothesized to be responsible for the inconsistent and highly variable antibody response seen over time in humans infected with KSHV. However, more studies are needed to determine the specific mechanisms and pathogenic purpose of this KSHV-mediated alteration of the Immunoglobulin repertoire, and how this may contribute to the phenomenon of seroreversion.

2.1 ∣. KSHV Serology Methodologies

With technological advances, serological assays to detect KSHV Abs have evolved. One of the most common detection methods is immunofluorescence assay (IFA) (Figure 1A). The frequently used target cells in IFA are KSHV-infected but EBV-negative B-lymphoma cell lines such as BCP-1, BC-3, and BCBL-1 [19, 38, 39]. These cells can be reactivated to express KSHV lytic and latent proteins with sodium butyrate or 12-O-tetradecanoylphorbol 13-acetate [40, 41]. The reactivated cells are then fixed and mounted onto glass slides, followed by sequential incubations with samples (e.g. sera), secondary Ab, fluorescent-conjugated tertiary Ab, and counterstain. The final stained slide is read with a fluorescence microscope. Although IFA is the fastest and cheapest of all the serology tests, it has the disadvantage of lacking a KSHV-negative parental cell line as a negative control. Additionally, IFA results are nonquantitative and assessment can be subjective. To overcome these limitations, research groups commonly rely on multiple reviewers and nonequivalent KSHV-negative B cell lines such as BJAB, or utilize KSHV-negative serum as negative controls [42, 43].

FIGURE 1 ∣.

Illustrative overview of the serological tools used to characterize KSHV-specific Ab responses. (A) Immunofluorescence assay (IFA) detects KSHV using fluorophore-tagged conjugated Abs. (B) Enzyme-linked immunosorbent assay (ELISA) involves antigen-coated plates and colorimetric detection of bound KSHV Abs via enzyme-conjugated secondary Abs. (C) Luminex-based multiplex bead assay utilizes antigen-coated beads to detect KSHV antibodies with high-throughput flow cytometry readout, while (D) Luciferase Immunoprecipitation System (LIPS) uses luciferase-tagged antigens incubated with patient sera and protein A/G beads, which are quantified via luminescence. (E) A protein microarray involves expressing and purifying KSHV open reading frames (ORFs), printed on slides and incubating with sera for proteome-scale Ab profiling. (F) Phage immunoprecipitation and sequencing (PhIP-seq) screens patient plasma against an extensive overlapping viral peptide library (VirScan). This is followed by magnetic pull-down of Ag: Ab complexes and multiplexed next-generation sequencing to profile viral Ab repertoires.

Another assay used to detect KSHV serology is the enzyme-linked immunosorbent assay (ELISA) (Figure 1B). ELISA was first developed in 1971 by Eva Engvall and Peter Perlman, and it has since been used to detect antibodies against a wide variety of antigens, including KSHV antigens [44-48]. A 96-well plate is first coated with the desired KSHV antigen [49] to perform the ELISA. Patient sera are added, allowing the KSHV-specific Abs to bind to the plate-bound antigen. An alkaline phosphatase or horseradish peroxidase-conjugated anti-human Ig Ab is subsequently added, followed by colorimetric development with the appropriate substrates. The absorbance from each well is then measured with microplate readers. Although ELISA is sensitive and quantitative, it requires purified viral proteins, which can often be a challenge for KSHV glycoproteins. As an alternative, the whole KSHV virus has been used as an antigen, but this approach cannot distinguish responses to specific viral proteins [49, 50].

One method to enable simultaneous detection of antibody response against multiple antigens is through multiplex bead-based assays [51, 52]. Microspheres (or beads) coated with specific KSHV proteins are combined with the serum sample, where the KSHV-specific Ab binds to their respective beads (Figure 1C). A fluorescent-conjugated secondary Ab is then introduced. The beads are analyzed using either a flow cytometer or a Luminex analyzer, which identifies the different bead sets (i.e., different antigens) and measures fluorescence intensity, which is directly proportional to the amount of Ab bound against each specific antigen. Multiplex-based immunoassays are generally more sensitive compared to ELISA-based assays, require smaller sample volumes, and have a lower cost per sample; however, they still require purified viral proteins as targets.

The Luciferase immunoprecipitation systems (LIPS) assay is another method used to measure Ab responses to a wide range of pathogens quantitatively (Figure 1D) [53, 54]. LIPS is known for its high sensitivity and ability to detect both very low and very high concentrations of analytes. LIPS involves generating a fusion protein between a target antigen and the reporter enzyme Renilla luciferase (Ruc), which is then transfected into mammalian cells for protein expression. The cell extracts of transfected cells, containing the Ruc-antigen fusion protein, are then mixed with the serum sample and immunoprecipitated with protein A/G beads. The antigen-specific Abs are then quantified by adding coelenterazine substrate, which oxidizes when bound to Ruc emitting blue light, and measuring the luminescence produced. Using LIPS, KSHV antigens K8.1, ORF73, ORF65, and vCyclin were tested, which demonstrated a sensitivity of 99% and a specificity of 100% for diagnosing KS patients, an accuracy comparable to ELISAs targeting K8.1 and ORF73 [55]. A separate study utilized LIPS to evaluate anti-KSHV Ab levels in an MSM cohort with or without KS, regardless of HIV infection status, and compared the results to ELISA to assess the utility of LIPS in detecting asymptomatic KSHV infection. They demonstrated that ELISA detected a higher KSHV infection rate (35.5%), which agreed with LIPS results for 81% of the samples [56].

A more comprehensive approach to studying antibody responses against the entire KSHV viral proteome is through a proteome microarray (Figure 1E) [51, 57]. This technique works by first purifying various KSHV proteins, which are then bound onto specialized glass slides, creating a microarray that includes multiple spots for each protein along with control spots. Biological samples, such as patient plasma, are added and followed by a fluorescently labeled secondary Ab. The signals on the slide are detected by a microarray reader. Although a proteome microarray provides a comprehensive readout, its high production cost and the need for specialized equipment limit the spread of its use.

Although proteome microarrays can detect KSHV-specific antibodies, they cannot determine the epitopes to which these Abs can bind. Recent advancements in epitope mapping technologies have led to the development of phage-immunoprecipitation and sequencing (PhIP-seq, Figure 1F) [58]. PhIP-seq involves the expression of an extensive library of short, overlapping 56-mers, peptides on T7 bacteriophages, with each bacteriophage encoding and expressing a single peptide [59]. The phage library is then incubated with the serum sample, followed by immunoprecipitation of the antibody-bound phages and next-generation sequencing of the extracted phage DNA. This approach allows for the profiling of antibody repertoires across complete proteomes without the need to purify each target protein or peptides. Still, more importantly, it only requires a very small sample volume. PhIP-seq has recently been used to identify anti-KSHV antibody repertoires in KS patients [25, 60]. The major limitations of PhIP-seq are that it is limited to detecting linear/quasi-linear and nonconformational epitopes and nonglycosylated targets.

Tools such as IFA, ELISA, LIPS, multiplex bead assays, and proteome microarrays have improved the diagnostic sensitivity and specificity, enabling robust detection and characterization of KSHV-specific antibodies. High-throughput approaches like PhIP-seq can identify novel antigens and epitopes that could be potential vaccine targets. Ongoing improvement in serological techniques for KSHV holds promise for enhancing our understanding of KSHV transmission, infection prevalence, and the risk of developing KSHV-related diseases among infected individuals.

2.2 ∣. KSHV Antigens Targeted by Humoral Immunity

Defining the KSHV antigens targeted by humoral immunity has been one of the objectives for various KSHV serology studies and can better inform KSHV vaccine designs. The most logical and critical viral proteins targeted by humoral immunity are the KSHV glycoproteins. KSHV encodes multiple glycoproteins, such as gB, gL, gH, gM, gN, and K8.1, which are essential for viral entry into the host cells, making them prime targets for nAbs and vaccines. However, there are limited studies on assessing anti-KSHV glycoproteins antibodies in infected individuals due to the challenges in generating and purifying full-length glycoproteins for in vitro assays.

Recently, by adsorbing nAbs with HEK293T cells expressing various KSHV glycoproteins, a study demonstrated the presence of KSHV-glycoprotein-specific antibodies in both asymptomatic and symptomatic KSHV-infected individuals [61]. Their findings showed that individuals could target multiple KSHV glycoproteins simultaneously, but the range and magnitude of these targeted responses vary widely between individuals. Interestingly, gH/gL complex and gB are the glycoproteins that are consistently targeted the most among individuals, which suggests these proteins as potential targets for vaccination. K8.1-specific antibodies have also been reported to be higher among KS patients compared to asymptomatic individuals, especially near their KS diagnosis, suggesting that monitoring certain KSHV-specific antibodies may provide some diagnostic insights into KS onset [17].

Because the immunodominant antibody profile in natural KSHV infection (e.g. strong K8.1 reactivity) is neither reliably protective nor consistently linked to disease severity, vaccine design should use natural immunogenicity as a starting point while refocusing B-cell responses onto conserved functional epitopes on gH/gL and gB, employing structure-guided multivalent display and adjuvants to increase potency and breadth, and optimizing Fc-mediated effector functions (ADCC/ADCP) with support from T-cell help. Although direct evidence for KSHV is lacking, experience with other enveloped viruses, such as SARS-CoV-2, shows that preexisting, vaccine-elicited nAbs can block viral entry at exposure. Such responses, ideally including mucosal compartments relevant to oral shedding, could reduce KSHV acquisition and early spread, whereas nAbs induced after infection are unlikely to be protective.

In addition to KSHV glycoproteins, numerous other KSHV proteins encoded by ORF4, ORF36, ORF38, ORF59, ORF61, and ORF65 have also been identified as targets of humoral responses [55, 57, 62, 63]. Although these Abs are not expected to have a significant impact on the disease pathogenesis, elevated levels of some of these antibodies might indicate higher KSHV plasma viral load or serve as prognostic markers for KS onset or development [64-68]. Interestingly, latency-related viral proteins such as LANA are also reported to be highly immunogenic despite being located mainly within the nucleus [67]. PhIP-seq data indicate that the central repeat region and C-terminus of LANA are more immunogenic than its relatively conserved N-terminus [60]. The same study also identified a 24-amino acid sequence within LANA that was the most immunodominant [60]. It is widely believed that these anti-LANA antibodies act as decoy viral antigens to divert the attention of humoral immunity away from its more sensitive proteins, such as gB. However, given the high prevalence of anti-LANA antibodies in infected individuals, some studies have used their presence to assess the infection status of individuals clinically and in various epidemiological studies [49, 57, 69-71]. Similarly, antibodies against another latency-related viral protein, vCyclin, were also detected in infected individuals and were used to discriminate between individuals with KS and MCD [51].

Taken together, these studies delineate a broad antigenic landscape for KSHV-specific humoral immunity while highlighting pronounced interindividual heterogeneity (Table 1). The magnitude, epitope specificity and breadth of responses vary substantially, even to the same viral proteins, likely shaped by host genetics, immune status and exposure histories (prior and concurrent infections). This variability complicates the definition of universal correlates of protection and instead argues for stratified, context-dependent biomarker development and vaccine design.

TABLE 1 ∣.

Comprehensive summary of KSHV antigens eliciting humoral immune responses, detection methods, response types, with supporting references and their key findings.

KSHV antigen (protein/epitope)	Type	Detection method	Key findings
gB (EC domain; conformational epitopes involved in membrane fusion)	nAb	Glycoprotein adsorption assay, ELISA, VLP vaccination	gB contributes significantly to total neutralizing activity. gB inclusion enhanced nAb titers, especially in combination with gH/gL and K8.1. Targeted in both asymptomatic and KS patients [24, 80].
gH/gL complex (conformational epitope)	nAb	Glycoprotein adsorption assay, VLP vaccination, ferritin nanoparticle platform	Strong and consistently detected nAb response. gH/gL is identified as the dominant neutralizing determinant in natural infection and vaccine models [24, 61, 80].
K8.1 (full-length protein, EC Ig-like domain, or linear epitopes)	IgG, nAb	ELISA (recombinant and nonrecombinant), LIPS, immunoblot, VLP assay, PhIP-seq	K8.1 vaccination induced KSHV nAbs, reduced viral loads and reactivation in mouse models. Robust IgG and neutralizing antibody (nAb) responses; elevated in KS patients [17, 25, 39, 49, 76, 80].
ORF4 (complement-inhibitory domain)	Complement-mediated neutralization	VLV immunization, antibody depletion and complement deposition, ELISA	VLV immunization elicited anti-ORF4 antibodies that mediate complement-dependent neutralization [63].
ORF4, ORF36/vPK, ORF38, ORF59, ORF61, ORF65, LANA, K8.1, ORF72/vCyclin (full-length proteins; arrayed ORFs, or linear epitopes)	IgG	Protein microarray, multiplex bead assay, LIPS, ELISA, PhIP-seq	Differential seroreactivity among KS patients, asymptomatic carriers, and controls. LANA: highly immunogenic, likely a decoy antigen. vCyclin: detected in infected individuals, differentiates KS vs. MCD. Seroreactivity may correlate with plasma KSHV viral load or lytic activation [25, 51, 53, 55, 57, 60, 62-68, 71].
gN, gM (predicted B-cell and CTL epitopes based on in silico screening)	Predicted IgG, nAb	Epitope prediction and structural docking (immunoinformatics)	Predicted to elicit IgG, including potential neutralizing antibody and T-cell responses; no experimental validation yet [87, 88, 90, 93-96].
Multiple KSHV epitopes (unspecified; whole-virus lysate and envelope-enriched fractions)	ADCC activity (IgG Fc)	ADCC reporter assay	FcγRIIIa-mediated ADCC. Detected, but not consistently in KSHV-seropositive individuals. Not predictive of KS disease presence or severity [29].
K8.1, ORF73 (full-protein IgG with unspecified epitopes)	Non-neutralizing maternal IgG	Longitudinal serological tracking (infant plasma)	Waning maternal IgG, which correlates with increased risk of KSHV acquisition in early childhood. Do not prevent infection but may delay seroconversion [23].

Abbreviations: EC, extracellular; VLP, virus-like particle.

Despite being known as a latency-prone virus that expresses a limited number of viral proteins, KSHV evidently can elicit humoral responses against a wide spectrum of its viral proteins in infected individuals regardless of their disease status. The magnitudes of these antibody responses vary between individuals but could be affected by contributing factors such as an individual's overall immune status and the presence of other coinfections. Understanding the dynamics of these responses could help to monitor or predict KSHV-related illnesses.

2.3 ∣. Targeting KSHV Antigens With Vaccines

Vaccines have been highly effective in targeting various pathogenic viruses, from the seasonal influenza to the recent SARS-CoV-2. The safety and efficacy of vaccines in preventing infection and the development of serious illnesses are indisputable. Although several FDA-approved virus-like particle (VLP)-based vaccines against oncogenic viruses such as HBV and human papillomavirus are commerically available, no vaccines are currently available against human herpesviruses. While there is active developmental progress toward an EBV vaccine, the progress toward an efficacious KSHV vaccine has been relatively limited.

The major obstacles for a KSHV vaccine are the lack of a suitable animal model, as the virus is known to only infect humans, and the tendency for KSHV to enter latency upon infection, which limits the availability of viral proteins for immune recognition. Several groups use murine gammaherpesvirus 68 (MHV-68) in mice and rhesus rhadinovirus (RRV) in macaques as surrogate KSHV vaccine models, leveraging their protein homology to KSHV and the availability of tractable animal systems [72-75]. However, the fundamental pathological differences between KSHV and these substitute viruses have prevented the direct translation of findings from these studies to KSHV pathogenesis and vaccine development. As attempts to overcome this issue, chimeric KSHV-MHV68 or KSHV-RRV viruses are being generated and used in vaccine studies [76-78].

As discussed earlier, the most common KSHV antigens targeted by various vaccine studies are its glycoproteins, gL, gH, gB, and K8.1. The efficacy of these vaccines in animal models has been extensively studied. For instance, gLgH administered either as a recombinant protein or expressed on a ferritin nanoparticle elicited nAbs in vaccinated mice. However, the highest neutralizing responses were only obtained in animals vaccinated with the wild-type (WT) gLgH proteins, demonstrating it as a strong candidate for KSHV vaccine development [79]. In another study, VLPs expressing gLgH, gB, and K8.1 also elicited neutralizing responses in vaccinated mice, but these responses were inferior to those achieved by UV-inactivated KSHV [80]. Interestingly, another study inoculated rabbits with a multivalent VLP, expressing gLgH, gB, and K8.1 simultaneously, and reported high neutralizing responses in vaccinated animals [81]. These studies indicate that vaccine efficacy depends not only on the antigen but also on how the antigen is presented to the immune cells. Preserving native trimer/heterodimer structures and controlling the meta-structure of epitope presentation, valency (number of epitopes), spacing (distance between epitopes), and orientation (3D alignment) can markedly improve B-cell receptor cross-linking and, in turn, the breadth and potency of the response. It is important to point out that none of these studies challenged their vaccinated animals with live KSHV; therefore, it will be difficult to determine the protective efficacy of these potential vaccines. Critically, the vaccine prime-viral challenge experiment is the gold standard in vaccinology. It will be extremely difficult to develop any KSHV vaccine without such experimental data, nor will it be possible to obtain regulatory approval to conduct clinical trials. To continue advancing toward an effective KSHV vaccine, there is an urgent need to develop a better animal model that can be susceptible to natural KSHV infection and generate the proper humoral and cellular immune responses after vaccination.

2.4 ∣. Application of Immunoinformatics to KSHV Vaccine Development

Immunoinformatics, a subdiscipline of bioinformatics that integrates immunology with computational methods, can potentially improve KSHV vaccine designs [82]. Identification of B and T cell epitopes is fundamental for designing better vaccines, and certain techniques, such as machine learning algorithms, sequence alignment, and structural modeling, are often used to predict linear and conformational B-cell epitopes that might elicit an Ab response. BepiPred [83, 84] and BEPI-BLAST [85] can predict B-cell epitopes based on amino acid properties and known antigenic sites. Homology modeling can further refine these predictions, especially for conformational epitopes that depend on the 3D protein structure. For example, DiscoTope predicts surface-exposed epitopes using protein structural data by applying a threshold score based on the relative solvent accessibility on the residues [86]. DiscoTope predictions are helpful in guiding experimental efforts to identify and validate such epitopes, especially in cases where experimental epitope mapping is challenging or expensive.

Since predicted epitopes often need validation for their antigenicities, tools such as VaxiJen [87] can analyze the epitopes to estimate their likelihood of eliciting an immune response primarily based on their amino acid sequences' physicochemical properties, and help prioritize potential epitopes for experimental validation. For instance, VaxiJen was recently employed in a study to predict antigenic epitopes from KSHV glycoproteins for designing a multiepitope vaccine [88]. Molecular docking simulations also predict how Abs might interact with KSHV antigens. Tools like ClusPro [89] simulate antigen–antibody interactions, while molecular dynamics simulations provide atomic-level insights into the stability and dynamics of these complexes [88, 90]. For instance, a study utilized ClusPro to model the interaction between the KSHV viral protein kinase (vPK) and the host deubiquitinase USP9X. The docking analysis predicted that vPK binds near the C1 domain of USP9X, suggesting a potential mechanism by which KSHV modulates host protein function during infection [91].

Recent studies have leveraged immunoinformatics to design multiepitope vaccines targeting KSHV. For instance, Chauhan and colleagues incorporated epitopes derived from key KSHV glycoproteins involved in viral entry and immune modulation in their vaccine design [88]. It included CD8+ (CTL), CD4+ (HTL), and IFN-γ-inducing KSHV epitopes that were identified using immunoinformatics tools such as NetCTL 1.2, and Net MHC II pan 3.2 servers with the IEDB consensus method [92, 93]. The IFN-γ-inducing epitopes were predicted using the IFNepitope server [94]. Sequences and physicochemical properties of KSHV epitopes were screened for conservation across strains, immunogenicity, nonallergenicity, and lack of overlap with human proteins to minimize autoimmune risk. Structural refinement of the 3D-modeled vaccine via homology modeling enhanced its stability and immune recognition potential. Furthermore, molecular docking and dynamics simulations confirmed stable interactions between the vaccine and Toll-like receptor 9 (TLR-9), which is known to recognize structural components of dsDNA viruses such as KSHV. The interaction pattern analysis of the multiepitope vaccine with TLR-9 showed a strong binding affinity, indicating that the vaccine construct could effectively engage TLR-9 and trigger an immune response.

In a complementary study, Hoque and colleagues focused on a vaccine targeting five KSHV envelope glycoproteins (gB, gM, gN, gH, and gL) critical for viral entry. Their design also incorporated multiple CTL, HTL, and B cell epitopes [90]. Unlike Chauhan and colleagues, Hoque's study evaluated the vaccine's interaction with TLR-4 through molecular docking and dynamics simulations, which is known to activate innate and adaptive immune responses. TLR-4 improved the vaccine by demonstrating its potential to form stable interactions with the immune receptor. Docking simulation was performed using the ClusPro docking server [95]. These stable interactions with TLR-4 were further supported by the PDBsum database and molecular mechanics with generalized Born and surface area solvation free energy decomposition analysis from HawkDock [96, 97], which suggests that the vaccine has a strong probability of inducing both innate and adaptive immune responses. For both studies, it is important to mention that the efficacies of the proposed chimeric multiepitope vaccines must be verified experimentally to determine their suitability for real-world use. Still, these in silico approaches offer a cost-effective foundation for guiding in vitro and in vivo vaccine development and epitope prioritization, which is crucial given the absence of an approved KSHV vaccine. In the absence of experimental challenge systems, insights from orthologous KSHV proteins and known host immune responses can guide the identification of optimal targets and protective anti-KSHV responses.

Future efforts should prioritize establishing a centralized KSHV database encompassing data from animal models and future clinical trials. Such a resource would include tested antigens, sequences, and immune responses, facilitating transparency, reproducibility, and efficient data sharing for targeted future experiments. While computational tools provide powerful insights, integrating them with experimental validation is essential to develop effective therapeutic and preventive strategies against KSHV.

3 ∣. Perspectives and Conclusion

It has been more than three decades since the discovery of KSHV. Although the KSHV field has made tremendous advancements toward our understanding of the virus and the diseases it causes, there is still much to discover and learn from. One of the areas that can be further improved is KSHV serology and the role of an effective humoral immune response. Currently, serological testing is the primary approach for assessing KSHV infection status. Serological testing is preferred over PCR mainly due to its relatively low cost and short turnaround time, and since KSHV DNA is not readily detectable in infected individuals due to viral latency. The cost of testing is a significant factor because most of the KSHV-infected populations are in SSA, with limited resources and access to healthcare; a rapid and sensitive point-of-care diagnostic test is much needed. The fastest KSHV serological tests, such as IFA and ELISA, take about one and a half hours to perform and cannot be readily done at point-of-care sites. In comparison, HIV serological rapid tests only take 15 min. A rapid and sensitive KSHV serological test will allow faster screening and diagnosis, so an infected individual can be advised on preventive actions to limit its spread. It might also reduce the chance of transmission through organ transplant, where timing is critical. Lastly, it is important to note that, with the rapid growth of technologies from bioinformatics to vaccine platforms, and intense investment and collaborations between academic, industrial, and regulatory agencies, it took less than a year to develop the SARS-CoV-2 vaccine. Similar effort is required to advance a KSHV vaccine. However, unlike SARS-CoV-2, developing a suitable animal model for KSHV infection will be paramount for the success of any potential KSHV vaccine and its use in the field.

Data Availability Statement

Data sharing is not applicable to this article as no data sets were generated or analyzed during the current study.