Cancer is a heterogeneous disease ^{[1][2][3][4][5][6]}[1,2,3,4,5,6] with a poor median survival rate [3]. According to the WHO, it is the second-leading cause of death worldwide ^[5][6][5,6], with breast cancer (BC) being the most common form diagnosed in females [7]. About 5–10% of patients diagnosed with BC exhibit its metastatic form [8]. Moreover, it is highly challenging to forecast the prognosis of the illness with high certainty [3]. BC is classified as invasive or non-invasive ^[9][10][9,10]. The invasive form includes infiltrating ductal carcinoma (IDC) and invasive lobular carcinoma (ILC), while the non-invasive form includes ductal carcinoma in situ (DCIS) and lobular carcinoma in situ (LCIS) [10]. BC is further categorised depending upon the expressing hormone receptor such as the estrogen receptor (ER+), the human epidermal growth receptor 2 (HER2+), the progesterone receptor (PR+) and triple-negative breast cancer (TNBC), i.e., ER, PR, and HER2-all negative [11]. TNBC makes up 10–30% instances of BC [12] and is distinguished by a higher rate of relapse, higher potential for metastasis, and a shorter overall survival [13]. Additionally, cases of male BC, which may be either congenital, developmental, or acquired [14], have recently increased by about 40%, outpacing female cases by 25% of the affected population and by 18% in terms of mortality ^[15][16][15,16], though accounting for fewer than 1% of total BC diagnoses ^{[14][17][18][19]}[14,17,18,19].

2. Vaccine Therapy in BC

2.1. Introduction

Vaccines have long protected humans from communicable and non-communicable diseases ^[20][102]. Therefore, conventionally, the word “vaccine” is usually related to the fight against infectious diseases. Vaccines exert their action by stimulating immune responses. This is achieved by inoculating a healthy individual with attenuated/detoxified bacteria, viruses, or extracted toxins ^[21][103]. The immune system works to keep living things in a state of equilibrium by the process called immune surveillance ^[22][104]. The method by which tumour cells circumvent the immune system has been extensively explored and has been successfully established over the past few years. Such efforts led to the conclusion that cancer immunoediting is the strategy employed by the tumour cells towards immune evasion ^[23][24][105,106]. It may be caused by TME-antigen-mediated antitumour immunological responses. A number of tools for cancer immunotherapy were developed, which include antibodies, peptides, proteins, nucleic acids, and immune-competent cells such as dendritic cells and T-cells ^[21][103]. Cancer immunotherapies are now considered the fourth treatment method ^[25][26][107,108], used either alone or in combination ^[27][60]. The therapy was initially utilised by William B. Coley in 1891, to treat sarcoma patients with Coley’s toxin ^[28][109]. The available vaccines for cancer immunotherapy can be divided into two basic types, the prophylactic and the therapeutic vaccines ^[29][30][110,111]. The former induces immunological memory by vaccinating healthy people ^[31][112], to prevent morbidity from a certain malignancy ^[32][113], and can be a cost-effective preventive measure ^[33][114]. The latter boosts immune systems in people detected with cancer ^[21][103]. The therapeutic cancer vaccine prevents the growth of advanced malignancies or relapsed tumours that are resistant to standard treatments ^[31][112]. Based on the structure and the content, the vaccines are further classified into the cell vaccines, the peptide vaccines and the nucleic acid vaccines ^[21][103]. The criteria necessary to be fulfilled to achieve vaccination requires a target antigen on tumour cells to stimulate the immune response, a vector to deliver the vaccine-derived antigen to the immune system, an adjuvant to boost immunological stimulation, and an appropriate monitoring tool ^[34][115]. These are discussed in detail in the subsequent sections.

2.2. Concepts in Designing of a Breast Cancer Vaccine (BCV)

2.2.1. Immunoediting

The immune system constantly changes as BC progresses. The process is called immunoediting, which comprises three steps: elimination, equilibrium, and escape. During elimination, the tumour cells stimulate the innate immune system (which along with the adaptive immune response can recognise and remove early altered tumour cells) through the activation of macrophages, natural killer cells (NK-cells), and dendritic cells (DCs), in turn activating the tumour-targeted T-lymphocytes. The equilibrium phase initiates in the case of a cancer subclone colony surviving the host’s immunity. This stage creates a delicate balance between cancer growth and the immune system’s defence function, making it difficult to totally remove the tumour cells, though their progression is severely limited ^[35][116]. However, it results in the formation of cancer cells with decreased immunogenicity, epigenetic alterations, and genetic instability ^[36][117], thus making them capable of escaping the immune detection and destruction ^[37][118]. Such cells that escape the immunological pressure finally enter the third stage of immunoediting, where the immune system barely puts any restraints on the progression of the modified tumour cells as in Figure 1 ^[38][119].

2.2.2. Immune Surveillance

Effector immune cells must directly recognise tumour antigens from tumour cells or indirectly from antigen-presenting cells (APCs), via the major histocompatibility complex (MHC) on the cell surface, to initiate an immune response. CD8+ and CD4+ T-cells are critical to the immunoediting process and help separate the non-self epitopes of the tumour cells expressed by MHC class I and MHC class II molecules from the normal self-antigens ^[39][40][120,121]. Tumour-specific antigens (TSAs) and tumour-associated antigens (TAAs) ^[41][122] are examples of tumour antigens. These include germ-line antigens, tissue differentiation antigens, and overexpressed antigens, exemplified by the melanoma-associated antigen, the carcino-embryonic antigen (CEA), and HER2 and MUC-1, respectively ^[42][123]. Many of the tumour antigens employed in immunotherapy are expressed in normal tissues as well. However, tumour cells overexpress these antigens ^[41][122].

2.2.3. Immune Suppression

Tumour cells successfully inhibit the host’s immune system, both locally and systemically, from evading immune surveillance ^[43][124]. It is well-established that there is predominance of the immunosuppressive effect with advancement of the disease. This results in a gradual transition from the elimination phase to the escape phase ^[44][125]. During this process, there is an alteration of the number of cells around TME and the lymph nodes in the vicinity of the tumour tissue. First are regulatory T-cells (Treg cells). There is enhancement in the proliferation of Treg cells brought about by transforming growth factor-β (TGF-β) ^[45][126], while cytotoxic T-lymphocytes (CTLs) are greatly reduced, as interleukin-2 preferably binds to Treg cells. The antitumour response is, thus, weakened by a reduction in the number of CTLs ^[46][127] and NK-cells ^[47][128] in TME (Figure 2). Treg cells function to downregulate the dendritic cell co-stimulatory markers, viz., CD80 and CD86, necessary for the priming of CTLs ^[48][129], which, in turn, function to eliminate any abnormal-phenotype-expressing cell. Second are tumour-associated macrophages (TAMs). TAMs release inhibitory cytokines including IL-10 and TGF-β to suppress CTL activity and the production of IL-12 ^[49][130]. Third are the myeloid-derived suppressor cells (MDSCs). During the shift, these cells start appearing in the peripheral blood as well ^[50][131]. Tumour cells also activate immunological checkpoint receptors such as cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed cell death receptor-1 (PCDR-1) ^[51][132]. PCDR-1 blocks programmed cell death ligand-1 (PCDL-1). Therefore, in patients with diverse malignant tumours, the tumour-infiltrating lymphocytes (TILs) and the tumour-specific T-lymphocytes show high amounts of PCDR-1. The involvement of PCDL-1/PCDR-1 by tumour cells prevents the elimination of T-cells, thereby causing their dysfunction and, ultimately, cell death. In contrast, the cytokines that create an immunosuppressive milieu help to further promote tumour growth ^[52][133].

2.2.4. Identification of the Antigen for BC Immunotherapy

Numerous tumour antigens, which are expressed in healthy cells but are overexpressed in tumour cells, are employed in BC immunotherapy. They include HER2, p53 (tumour protein 53), MUC1, carcinoembryonic antigen (CEA), telomerase reverse transcriptase (hTERT), and carbohydrate antigens ^[53][78]. Due to their widespread expression in the majority of tumour types, some of these antigens are known as universal tumour antigens. An example of this class is hTERT ^[54][134]. All the potential antigens that are used in the creation of vaccines for the management of BC are briefly discussed in the following section.

Human Epidermal Growth (HER2) Receptor 2

HER2 is a tyrosine kinase that regulates cell proliferation and survival. The oncogene for HER2 is located on chromosome number 17q12. In cases where BC is HER2-positive, an amplification of 15–20% in the expression of HER2 is found. This is responsible for triggering the growth and progression of tumour cells ^[55][135]. HER2 is linked to a more severe form, a higher risk of recurrence, and a higher mortality rate. The first drug to be established as a HER2 blocker was Trastuzumab ^[56][136], which has been validated for the treatment of patients with HER2+ BC ^[57][58][137,138].

Tumour Suppressor p53 Protein (p53)

The gene encoding p53 is located on chromosome number 17 ^[59][139]. It plays a vital role in the maintenance of DNA integrity and the prevention of cancer. Under normal physiological conditions, when a cell’s DNA is damaged, there is an induction of the p53 protein that causes arrest of the cell cycle. This allows cells to repair themselves, but, if the damage is too severe, the cells apoptise and are rejected. In a variety of cancer forms, mutations in the p53 gene (mtp53) takes place ^[60][140]. However, mtp53 is less frequently found in BC ^[61][141], though it has high significance in the diagnosis and prognosis of TNBC, with almost 70%–80% of cases displaying mtp53 ^[62][142].

Mucin1 (MUC1)

The MUC1 gene is encoded by chromosome 1q21. It is a high molecular weight transmembrane glycoprotein that functions as a physical barrier to protect the epithelial layer of cells from environmental exposure. It forms the epithelial lining of the respiratory and GI tracts, mammary glands, pancreas, liver, and kidneys. MUC1 is a poly-morphic type I member of the mucin family. It is overexpressed in about 90% of human BC due to genetic alterations and the dysregulation of transcription ^[63][64][143,144]. MUC1 is also aberrantly glycosylated ^[65][145], which exposes various antigens, thereby generating a new set of antibodies that can prove beneficial in the diagnosis of cancer ^[66][146].

Carcinoembryonic Antigen (CEA)

The CEA is a glycoprotein that is encoded by human chromosome 19q13.2. This antigen is an important serum biomarker for the detection of cancer and plays a vital role in the prognosis and diagnosis of BC. It is found elevated in patients with metastatic BC, especially with bone metastasis ^[67][68][147,148]. Several studies suggested the significance of CEA and CA15-3 levels in predicting BC patients who can be operated on at an early stage ^[69][149].

Human Telomerase Reverse Transcriptase (h-TERT)

The gene encoding h-TERT, a catalytic subunit of the telomerase, is encoded on chromosome 5p15.33. This ribonucleoproteic enzyme is responsible for synthesising telomeres, which, in turn, maintain the chromosomal length. The ultimate result is cellular immortalisation ^[70][150]. Studies suggested their vital role in the development of cancer. It was found that in most forms of cancer, the reactivation of telomerase takes place in a dependent or an independent manner. The enzyme ensures the stability of chromosomes, thus bypassing senescence. TERTp is the promoter region of TERT. Mutations in the TERT gene support carcinogenesis with variable frequencies, which takes place through the healing of the telomere’s length, thus expanding the life of cells. TERTp was established as a tool to characterizes the type of cancer ^[71][151] and, thus, is helpful in the diagnosis and prognosis of the disease ^[72][152].

2.3. Design Approaches of a BCV

The optimisation of vaccine schedules and administration methods is a component of a vaccination strategy. Based upon the platforms and formulations, BCVs can be broadly classified as peptide- and protein-based ^[73][153], carbohydrate antigen-based, whole-cell-based, gene-based ^[74][154], and fusion-based vaccines ^[75][155]. However, irrespective of the type, all vaccines are dependent on the autologous immune system recognising a specific antigen to exert a therapeutic effect. Moreover, the use of an adjuvant is essential, as it helps to increase the antigen immunogenicity, thereby controlling the immune response ^[76][156].

2.3.1. Peptide- and Protein-Based Vaccine (PV)

In this class of BCVs, the MHC class I restricted peptide epitopes are used to stimulate the immune response against a tumour antigen as shown in Figure 3 ^[77][157]. The injected peptide stimulates immune effector cells to find and kill cancer cells ^[78][158]. Some short amino acid peptides are preferably used, as they are cheap, stable, and easy to synthesise and modify and display low immunogenicity ^[79][159]. However, a vaccination cannot be given to patients with a non-common human leukocyte antigen type, since each peptide is confined to a specific HLA subtype. MHC class I binding peptides poorly stimulate CD4+ helper T-cells and also, in turn, limit their ability to activate CD8+ cytotoxic T-cells, resulting in a transitory immunological response. According to Pallerla et al., long peptides are capable of containing many MHC class I and class II epitopes, thus helping to partially overcome this difficulty. Such peptides containing 23–45 amino acids may improve T-cell activation through processing and presentation ^[74][154]. The success of the peptide-based BCV can be seen from its entry into phase I/II of clinical trials ^[80][160]. The entire tumour antigen protein or a truncated portion of it, wherein the sequence of amino acids is substantially longer than that of peptides, is used to create protein-based vaccines ^[81][161]. It is not HLA-restricted and allows for the absorption, processing, and presentation of a variety of MHC class I and class II peptide epitopes ^[82][162]. However, the presenting method may be less effective, and the lack of a precise marker makes it difficult to estimate how well such vaccine types would work ^[83][163]. A number of clinical trials are currently in the pipeline to test the efficacy of vaccines pertaining to BC ^[82][162].

2.3.2. Carbohydrate Antigen-Based Vaccine (CAV)

Immune cells can discriminate between improperly expressed carbohydrate antigens in tumour cells. This makes the carbohydrate antigen a prime candidate for inclusion in cancer vaccination. An example includes the expression of a unique disaccharide carbohydrate, Sialy-Tn (STn), on the cell surface of cancer cells, including BC cells, which is connected to MUC-1 ^[84][164]. According to Munkley (2016), immunisation with STn results in tumour regression and prolongation of survival time. Thus, it could be beneficial in the development of a cancer vaccine ^[85][165].

2.3.3. Whole Tumour Cell-Based Vaccine (WTCV)

One of the conventional approaches in the development of a cancer vaccine is the stimulation of the immune response by the use of whole tumour cells or the products obtained from tumour cell lysis. A WTCV induces a polyvalent immune response, since it is based on a pool of unknown antigens created by autologous or allogeneic tumour cells ^[86][166]. Sometimes, the enhancement of the antigen-presenting ability of the WTCV is achieved through engineered tumour cells that are capable of releasing cytokines or expressing co-stimulatory molecules. The drawback of the WTCV is that it contains endogenous cellular antigens, which can result in an autoimmune response. A systematic procedure for creating the WTCV is, however, lacking ^[87][167].

2.3.4. Dendritic Cell-Based Vaccine (DCV)

Upon migration into the lymph nodes, a diverse population of APCs, called DCs, effectively absorb antigens to process and present them to CD4+ and CD8+ T-cells. DCs can also stimulate NK-cells and B-cells. DC-based vaccines typically use ex vivo generated DCs that were transfected to express tumour antigens or are loaded with tumour antigens. In a review by Butterfield et al., the use of antigens, such as complex tumour lysates and several MHC class I and class II peptides, and monocytes and CD34+ progenitor cells for the purpose of DCV preparation was discussed ^[88][57]. The technical challenge that arises while developing DC-based vaccines was because of the distinct process of ex vivo DC maturation ^[89][168].

2.3.5. Gene-Based Vaccine

The strategy for a gene-based vaccine involves a plasmid that carries the DNA encoding the cancer antigen. This form of vaccine can be used to activate both a non-specific innate immunity and an adoptive immunity specific to an antigen. Due to its simplicity, safety, and cost-effectiveness, this approach is regarded as one of the most practicable methods for cancer immunotherapy. However, it suffers from limitations such as a lack of plasmid uptake and ineffective antigen expression, thus presenting insufficient immunogenicity ^[82][162]. To overcome this demerit, various approaches were undertaken. One of the approaches was to make the vaccine self-replicating. This utilised the RNA replicase encoding gene ^[90][169]. RNA-based medications have the potential to be effective pharmacological regulators against cancer cells by altering the expression of particular proteins. These characteristics help to increase specificity and reduce the chance of off-target impacts ^[73][153].

2.3.6. Fusion Vaccine

This strategy employs the fusion of autologous DCs and autologous whole tumour cells. It involves the cytoplasm of both cell types to be fused together without their nuclei doing so. This preserves their ability to function as individual cells. With the fusion, the formed vaccine can express and process a wide range of recognised and unrecognised tumour antigens ^[91][170]. Studies and trials found a favourable response by patients with metastatic BC towards this fusion vaccine, in terms of its potent antitumour effects ^[74][92][154,171].

2.4. Adjuvants Used in Design of BCVS

Adjuvants are combined with the antigen and are essential in the case of low immunogenicity. They help to increase the immunogenicity of the antigen and thus, trigger, the immune response, particularly in the elderly ^[76][156]. Most adjuvants work by decreasing antigen release, encouraging antigen absorption and presentation by APCs, and boosting the growth of DCs and macrophages ^[93][172]. Traditional adjuvants, such as alum, mostly stimulate type-2 T-helper cell-dependent humoral immunity in prophylactic vaccinations for infectious diseases rather than type-1 T-helper cell responses that directly destroy tumour cells ^[94][173]. A common adjuvant in BCV is a secreted cytokine, called granulocyte-macrophage colony-stimulating factor (GM-CSF). It was demonstrated to promote the proliferation and activation of DCs as well as the maturation of myeloid cells such as granulocytes and macrophages ^[95][96][174,175]. Clinical trials of many GM-CSF-containing BCVs revealed measurable immune responses. The local administration of GM-CSF to melanoma patients increases the probability of the antigen immune response after vaccination. Studies found GM-CSF suppresses T-cell responses and produces inhibitory MDSCs. However, elaborative research is needed to discover the role of GM-CSF as an adjuvant for cancer vaccines. DNA-based cancer vaccines also use recombinant viral vector adjuvants (Figure 4). Recombinant viral vectors, which commonly carry antigens, contain different levels of pattern recognition receptor (PRR) and toll-like receptor (TLR) ligands that activate DCs and boost immune response. TLR agonists activate CD8+ T-cells and prevent T-cell exhaustion. The vectors contain other sequences that can compete with the targeted antigen motif, which is the main drawback of this adjuvant. However, adjuvant effects vary depending on vaccination formulation, targeted tumour antigens, immunisation schedule, and mode of administration, making adjuvant comparisons difficult ^[97][176]. Moreover, adjuvant optimisation tests for BCV are crucial.

2.5. Routes of BCV Administration

A good vaccine for cancer should be capable of efficiently transferring antigens to autologous APCs. For this purpose, various strategies are preferred. HER2 peptide-based vaccines are usually intradermally administered and exhibit an enhanced rate of response. This is probably due to the widespread network of DCs. Low intradermal peptide doses are safe and trigger specific responses by antigen T-cells in most healthy human subjects ^{[98][99][100]}[177,178,179]. Several BCVs were tested for their immune response induction efficiency via subcutaneous injection ^[101][180]. However, a large dose of antigen at the site of injection may cause severe reaction and sporadic sterile abscesses, which may require vaccine discontinuation or a dosage reduction ^[96][175]. Vectors or plasmids are injected through various routes in the case of the administration of a DNA-based vaccine, of which the intramuscular route of administration was found to display the most effective immune response ^[102][181]. Some DC-based immunisations must be given intravenously to directly stimulate lymph node T-cells ^[77][157].

2.6. Clinical Trials of BCVs

In preliminary studies, certain BCVs were successful in eliciting discernible immune responses and showing good tolerance. However, most of them showed appreciable clinical advantages in the ensuing Phase 3 trials. Recently, the NeuVax^TM vaccine (developed by Galena Biopharma, a US-based biotechnology company), which is administered with Leukine^® for the disease condition of BC with intermediate to low HER2 expression, was granted a Special Protocol Assessment for its Phase 3 trial (Prevention of Recurrence in Early-Stage, Node-Positive Breast Cancer) ^[103][182]. The Theratope^® and Enhanzyn™ vaccines are some of the vaccines that were withdrawn from the clinical trial enrolment ^[104][183]. A brief summary of the vaccines in clinical trials is listed in Table 1.

NCT Number	Antigens/Biological	Clinical Phase
NCT00854789	E75 and GM-CSF	I
NCT00892567	Her-2/neu; CEA and CTA	I
NCT02019524	E39 and J65 peptides	I
NCT04270149	ESR1 peptide vaccine	I
NCT04521764	Helicobacter pylori neutrophil-activating protein	I
NCT00343109	HER-2/neu	II
NCT02348320	Personalised polyepitope DNA vaccine	I
NCT02018458	LA TNBC; ER+/HER-BC	I/II
NCT04348747	Anti-HER2/HER3 DC vaccine; Pembrolizumab	II
NCT02061423	HER-2 pulsed DC vaccine	I
NCT01730118	AdHER-2/neu DC vaccine	I
NCT00524277	HER2-derived peptide GP2; GM-CSF	II
NCT01479244	HER2-derived peptide E75; GM-CSF	I/II
NCT01570036	HER2-derived peptide E75; GM-CSF; Trastuzumab	II
NCT00140738	HER; AS 15	I/II
NCT02061332	HER; DC vaccine	II
NCT00399529	HER2; GM-CSF; Cyclophosphamide; Trastuzumab	II
NCT01479244	HER2-derived peptide E75; GM-CSF	III

2.7. Combinational Therapy of BCVs

Immune checkpoint blockers (ICB) have changed the approach towards the treatment of cancer. As far as BC is concerned, ICBs were already proven to be effective in treating metastatic TNBC ^[105][184]. The addition of ICB to Trastuzumab, however, was linked to additional side events and did not provide a clinically substantial improvement in the progression-free survival for HER2-positive metastatic BC ^[106][185]. Combining the vaccination with ICB to combat cancer tolerance is a trending research approach in the field ^[107][186]. As previously stated, ICB blocks inhibitory receptors such as PCDR-1/PCDL-1 and CTLA-4 to enable the effector immune cells to kill tumour cells. According to certain preclinical research, when T-cells are activated by tumour vaccines, the inhibitory receptor expression on the cell surface also increases. One of the underlying reasons is the enhanced interferon-γ (IFN-γ), which is released by tumour-specific T-cells. IFN-γ up regulates the expression of PCDL-1 on the tumour cells and APCs. PCDL-1 initially helps to prevent the body’s immune responses from being too amplified ^[108][109][187,188]. Therefore, the immunosuppressive impact that reduces the antitumour immunity elicited by vaccines is likely to be relieved by an injection of ICB ^[110][189]. A promising approach that has the potential to improve and lengthen the course of the immune response and effectively produce considerable clinical benefits is the combination of the BC vaccination with ICB. Additionally, combining cancer vaccinations with recognised medicines could also increase efficacy. The research suggests that some HER2-derived peptide vaccines ^[111][190] and anti-HER2 monoclonal antibodies may function synergistically ^[112][191]. Studies show a connection between chemotherapy/radiation therapy and immune-related cell death. When these medicines are applied in conjunction with cancer vaccines, it might create a long-lasting immune response. Consistently, it would be worth investigating the effects of integrating cancer vaccination with chemotherapy ^[113][114][192,193], hormone therapy ^[115][116][194,195], targeted therapy ^[117][118][196,197], and radiation therapy ^[119][120][198,199].

2.8. Challenges Faced during the Course of Development of BCVs

Despite the several advantages associated with cancer vaccines, there are numerous challenges accompanying cancer vaccination strategies. Firstly, the development of tolerance towards the antigen, which results in lowered immune response. Secondly, the heterogenicity of the tumour type brings in some diverse intrinsic and extrinsic pressures, which, in turn, result in a detrimental effect on the antitumour vaccination processes. In addition, some genetic and non-genetic mechanisms play a role in defining the heterogenicity of cancerous cells, thus affecting the immune response. Moreover, the immune invasion mechanism was found to be a major hurdle in the efficacy of a vaccine. This requires potential alternative strategies to improve antitumour immunity ^[121][200]. Some developed vaccines were found to display poor immunogenicity when used alone. Therefore, the next generation of adjuvants may be used ^[122][201]. A key point during the development of an anticancer vaccine is the aftermath, in the form of immunological responses. Thus, it is necessary to recognise the immune modulatory pathway, and test and validate the disease-specific cancer vaccination ^[123][202]. Cancer immune interaction is also governed by the concomitant use of a drug.