An overview of vaccine production against shrimp White Spot Syndrome Virus, effects and the possible impact of this technology in Ecuador

: Although aquaculture in Ecuador has a high economic and socio-cultural importance, pathogenic microorganisms affect the development and vitality of crustaceans, fish, and mollusks, reducing their production yields. Among these pathogens, White Spot Syndrome Virus (WSSV) is an invertebrate virus that induces high mortality, generating severe economic losses due to its wide geographical distribution and high infection rate finding the most significant devastation worldwide in the shrimp sector. Although several strategies are described to fight against WSSV, this study points to an updated overview of vaccines used against this virus, including types, effects and large-scale production ways. Thus, this research supplies an analysis of possible treatments based on vaccination to combat the WSSV caused-disease that significantly impacts the aquaculture economy and could be helpful to those working in this field.


Introduction
In the last 50 years aquaculture industry in Ecuador has become one of the most critical sectors for the domestic economy since more than 40% of Ecuadorian exports are related to this income source 1 . During 2021-2022, shrimp production reached in the country 848,000 MT with a profit of 5323.30 million dollars 2 , making the country one of the largest shrimp exporters worldwide. The European Union (EU), Russia, the United States and China are currently the four main destinations for Ecuadorian shrimp exports 3 . However, diverse types of diseases caused by DNA and RNA viruses significantly affect shrimp production. Three types of viruses have been identified that drastically affect farmed shrimps in the country: Infectious Hypodermal and Haematopoietic Necrosis Virus (IHHNV), Taura Syndrome Virus (TSV) and White Spot Syndrome Virus (WSSV) 4,5 . All these three viruses in Ecuador caused significant economic and social losses. The primary example is the appearance of WSSV in 1999, which caused a 50% decrease in production and exports during the first years of the incidence, with the subsequent jobs lost in multiple families 4, 6,7 .
WSSV can infect many aquatic crustaceans, especially decapods, such as marine brackish and freshwater shrimps, sea crabs, crayfish and lobsters 6 . However, neither does it cause problems for human health or food safety nor affects human shrimp consumption while causing a detrimental effect on shrimp farmers' production 8 .
World Organisation for Animal Health (WOAH) included White Spot Syndrome Virus in a list of infectious diseases that are considered to be of national socioeconomic and/or public health significance and whose effects on international trade in animals and animal products are not negligible 6,7 .
Several approaches have been used to combat the in-cidence of infectious diseases, including antivirals, prebiotics, plant extracts-based drugs and antibiotics [9][10][11][12] . Although several strategies exist to combat WSSV [13][14][15] , this study provides an up-to-date overview of production, effects and types of vaccines against WSSV in shrimp. Thus, this research supplies an analysis of potential possible treatments and new tools to fight against this disease that significantly impacts the aquaculture economy, not only in our country.

Shrimp immune system and response to vaccines
The innate immune system is pronounced in shrimps to protect them from external agents and pathogenic microorganisms 16 . Crustaceans are generally known not to have a specific immune system 17 , which precludes the use of conventional vaccines to treat pathogens. According to Afsharnasab (2014), crustaceans' immune system comprises three defense mechanisms, all needed to defend themselves, as depicted in Figure 1.
The first is the cuticle and skin's physical and chemical defense system encompassing secretions [18][19][20] . This system is inefficient in protecting the organism from all pathogens because most crustaceans have an open circulatory system. The second line of defense is the cellular one. In the crustacean's world, these cells are called hemocytes and are composed of hyaline, granular and semi-granular cells. Each of them has a significant role in disease prevention. The last one to mention is the humoral defense 21 .
Innate immunity is triggered when pathogens are detected by host proteins, such as antimicrobial, coagulation and pattern recognition proteins, which, in turn, activate humoral or cellular effector mechanisms to destroy invading pathogens 22 . Figure 2 shows the 3D structure of Beta 1,3-Glucan Binding Protein (BGBP) found in plasma, which serves as a protein recognizer in the arthropod immune system 17 . This is in conjunction with the transglutaminase enzyme which is released by hemocytes in the presence of pathogens through receptors 25 . Lectin protein is also represented in the immune system with an antiviral function recognizing WSSV proteins 25,26 . In addition, antimicrobial peptides like Stilicin have antibacterial activity when interacting with the LPS endotoxin of gram-negative and show vigorous activity against filamentous fungi 27 .
On the other hand, Alpha 2 macroglobulin, a high molecular mass proteinase, generates opsonization activities against invading pathogens by mediating endocytosis 28 . Penisidins, other essential proteins, are active against Gram-positive bacteria by binding them, causing agglutination, and additionally, in high concentrations, have a good effect against fungi 29 . These are some of the main proteins responsible for humoral immunity 31 .
Studies show an alternative memory immune response; however, there are no T cells, B cells or major histocompatibility complex (MHC) molecules 30 in shrimps. Recent experimental data from shrimp and other arthropods have shown that invertebrates own an alternative memory type of immune response. This memory-like peculiarity is called resistant priming 22,31 . With this mechanism, shrimps could improve their defenses after initial pathogenic exposure and then generate better protection after subsequent infections with the same or a different pathogen.
Laboratory tests have shown that vaccinated shrimp and crayfish have improved survival rates following exposure to WSSV 32 . Penaeus japonicus, which survived natural and experimental WSSV infections, initially resisted subsequent WSSV exposure. However, these results were not replicated under different conditions -such as temperature, country or type of shrimp 33 . But it is not a treatment that can be applied overnight, mainly because of the unique adaptive immunity of shrimps 34-36 .

White spot syndrome virus
Several virus families affect invertebrates; some include DNA viruses 37 such as Nimaviridae, Parvoviridae, Baculoviridae and Iridoviridae 38,39 , which has the most significant impact on shrimp farming.
This article focuses on vaccines against the White Spot Syndrome Virus (WSSV), one of the most lethal arthropods viruses worldwide, with a mortality and infectivity rate in shrimp of up to 100%, significantly affecting the larval stage generating large economic losses 4,40 .
WSSV is a double-stranded DNA virus with an approximate genome size of 290 to 300 kb, which makes it one of the most complex viruses infecting shrimp 4,40 . Most of its putative translated gene products have no homology with other virus proteins or host cells. Because of this peculiar Figure 1. Shrimp defense mechanisms against potential pathogens. The first defense mechanism is the cuticle, and the second consists of cellular defense, including cytotoxicity, coagulation, encapsulation, phagocytosis, melanization, apoptosis and modulation. The third humoral defense mechanism is based in the action of hydrolytic enzymes, agglutinins, coagulation proteins, antimicrobial peptides, oxygen and nitrogen free radicals, and effectors. All three mechanisms act together to eliminate foreign agents 13,17,23,24 .
feature, the International Committee on Taxonomy of Viruses (ICTV) classified WSSV in its own family: Nimaviridae, within a unique genus Whispovirus 40,41 .

Impact of WSSV virus on Ecuador's Economy
It is believed that WSSV entered Ecuador by importing contaminated larvae from Panama, spreading to the natural environment and later contaminating all farms. The virus was established between 1999 and 2000 42 , causing great economic losses for the producer and the country itself. The National Institute of Fisheries (NIF), attached to the Ministry of Agriculture, Livestock, Aquaculture and Fisheries (MA-LAF), carries out annual tests 43 in several shrimp farms to determine the presence of different diseases using molecular tests.
There is evidence from the early 1990s that exports generated revenues for the country of around 3.5% of gross domestic product (GDP) on average, rising to almost 4.5% of GDP in 1997, 1998 and 1999 1 . After these years, the White Spot Syndrome epidemic broke out all over the world, and shrimp exports dropped to 2% in 2000 and to less than 1.5% in 2001. The shrimp industry and the Ecuadorian economy suffered significant damage until 2010, when a new increase in the export earnings of this product began reaching higher levels than before due to the control of the shrimp farms before the disease, as seen in Figure  3. Currently, the government conducts annual monitoring that allows the early detection of diseases. It is necessary to point out, that there is no protocol to deal with this virus in case it emerges again 1,44,45 .
In August/September 2019, shrimp exports from Ecuador to China significantly dropped due to the presence of WSSV in the shipments; China is the leading importer of Ecuadorian shrimp worldwide. Therefore this problem ge-nerated a significant loss in annual profit, affected subsequent trades and caused the suspension of shrimp exports to China from various Ecuadorian companies 48 .
As a result of the last infectious trade between Ecuador and China in 2019 a, better product management, constant monitoring and an adequate prevention protocol allowed to control the virus outbreak and thus not generate problems as such, increasing exports to that country 49 .

Major vaccines designed to combat infectious diseases in shrimp
Disease-fighting protocol development in shrimp involves the characterization of immune system effectors and understanding defense reactions to potentially lethal pathogens, considering that pathogen-host interactions are constantly changing 49 .
Vaccination is a defense mechanism used to enhance the shrimp immune system, which has been studied since the 1990s 9,50,51 . WSSV is one of the most serious pathogens affecting shrimp farming worldwide, so vaccine supplies constitute a significant protective benefit for the shrimp host.
Different vaccines have also been developed to combat the WSSV based on both the capsid and the core proteins, but also virus fragments or even completely inactivated viruses have been used 52,53 . The technologies currently employed are nanoparticles as vectors and gene silencing to prevent virus proteins from binding to shrimp cells generating an efficient immune response 53 .
In aquaculture, 3 types of vaccines are commonly used. Live Attenuated Vaccines include a suspension of a live attenuated pathogen that generates a response that does not allow excessive replication despite the ability to multiply in the host 54 . Live vaccines cause an asymptomatic, self-limiting infection. Therefore, the host immune system resem-An overview of vaccine production against shrimp White Spot Syndrome Virus, effects and the possible impact of this technology in Ecuador bles natural infections in a controlled system 55 .
The second type of vaccine is recombinant vaccines which refer to immunogenic proteins or purified epitopes obtained from the pathogens or carriers. These can also be composed of the virus's DNA or dsRNA, as in the Recombinant Infectious Haematopoietic Tissue Necrosis Vaccines 56 . This type of vaccine has been one of the busiest in the last decade, primarily to molecular advances and studies of recombinant virus subunits 57 . More than 40 WSSV structural proteins have been identified 22 and used to manufacture efficient recombinant vaccines. Among these proteins are VP19, VP24, VP26, VP28, Vp36, VP36B, Vp37, VP39. Proteins VP19, VP24, Vp36B and VP39 are found on the WSSV envelope 22,58 . VP15, VP26 and VP36 are proteins found in nucleocapsid 22,59 . Because the structural proteins are the first to act with the host 60 , those are considered the basis for neutralization strategies or the most likely candidates for vaccine development.
Particular studies have also shown that shrimp vaccinated with recombinant plasmids or microorganisms carrying a gene for the most studied WSSV coat protein (vp28) could efficiently protect shrimp against WSSV infection 57,[61][62][63][64] .
The third type is the inactivated virus vaccine, prepared from the suspension of completely killed cells of bacteria, viruses, or fungi. This type of vaccine has been successful against different disease-caused agents, such as Vibrio anguillarum, Vibrio salmonsida, also used in white shrimp, with good results. These vaccines are produced using chemical and physical (heat and radiation) inactivation methods. The most critical step in the production of such vaccines is inac- Lastly, there is another type of vaccine that is not widely used in aquaculture but is commonly used in the veterinary and human area., that is the case of synthetic vaccines manufactured from polypeptides that simulate the primary sequence of antigenic amino acids. Its function is very similar to that which occurs with inactivated viruses 54 . Table  1 shows the type of vaccine, composition, how the active ingredient was obtained and the survival rate for each study.
According to the gathered data, the vaccines with the highest incidence were the envelope protein vaccines VP28 [54][55][56]65 . This protein plays a role in interacting with the host cell surface 64 , which has been the most studied since the virus first appeared in 1992 62,63 . VP28 is one of the most critical targets for vaccine manufacture, as it is one of the main WSSV coat proteins and acts as a binding protein, allowing the virus to combine with the shrimp cells and letting it join the cytoplasm 57 .
The combination of this protein and others, such as Vp37, an envelope protein that facilitates infection, does not reduce the infection rate. Still, it does allow an improvement in the time of resistance to WSSV. Another of the mixtures is with VP24, as it is the only infection protein that has been shown to interact with the host polymeric immunoglobulin receptor protein (MjpIgR), which can mediate WSSV infection, generating good resistance results 57,66 .

Designed specific vaccines against WSSV used in shrimp production
The primary purpose of vaccines is to stimulate the  An overview of vaccine production against shrimp White Spot Syndrome Virus, effects and the possible impact of this technology in Ecuador shrimp's immune system and generate a defense response against WSSV to prevent virus scape and thus reduce its replication and spread 101 . These cell responses against WSSV are given in different ways. Two of them stimulate the cell response by: 1-the presence of biomolecules belonging to the virus and 2-molecules that interfere with the receptors where the virus assembles to the host cell. On the other hand, genetic modifications that provide a protective response by not generating interactions in the cells with the virus 102 also result in good practice.
Among the revised papers, 34 deal with recombinant vaccines, the most used ones based on recombinant proteins from the structural parts of WSSV. The combination of 2 or more structural recombinant proteins 5 generates a higher protection rate against this virus 61,83 . The revised reports also determined that the main type of vaccine is composed of the subunit-recombinant, polysaccharide, and combined subunit vaccines. According to Figure 4, the vaccines mentioned above showed a protection percentage of 73.91%, while other treatments related to both; synthetic or inactivated virus vaccines reached lower protection percentages of only 6.52% and 19.57%, respectively. In this figure is also noticed that the most frequent active principle is the recombinant vp28 protein, reaching 21.74% of incidence.
Experimental conditions are very important in reaching a good performance of any vaccine treatment against WSSV since protection results could change from one experiment to another according to the experimental conditions. Some parameters to take under consideration in this experiment are a) the type of shrimp, b) the form of virus replication referring specifically to the animal used, c) the region in which the study was carried out where the environmental parameters varied and d) the variation of virus infection that can reach mortality levels up to 100% 103,104 . Interestingly, in some research reports, there was no total mortality, mainly due to the resistance some arthropods can generate against WSSV 105 .
Also, administration methods at the production level deal with the efficiency in the vaccination methods 99,100 . It is worth noting that the most common method of vaccine administration is intramuscular administration, with a frequency of 48%, followed by oral administration at 42% and finally by immersion at 10%. However, oral vaccine administration is the best and most studied method at the industrial level.
The effect on the immune system produced by the vaccine in shrimp is calculated by the efficiency of the treatment against WSSV, demonstrated by the number of vaccinated animals that survived exposure to the virus; the treatment with the highest efficiency and best protective effect was the intramuscular administration. The treatment with the highest efficiency and best protective effect was the intramuscular route, with 18% of treatments having a survival rate of more than 75%; the oral way had an efficiency of 10% for medicines with a survival rate of more than 75%, and the immersion route had a frequency of 6% for treatments with a survival rate of more than 75% ( Figure 5).
It was determined that, in general, the efficiency of the vaccine is between 50% and 75% of shrimp survival rate reaching 52% of frequency in the studied articles, followed by others that reached an effectiveness of more than 75% having 34% of frequency, allowing to assert that vaccination is an effective treatment against the virus.
Detailing the efficiency depending on the vaccine composition, it was determined that gamma-inactivated virus is the most effective, reaching a 6% frequency in treatments with a survival rate of 75%, followed by vaccines made with recombinant VP28 protein, which reached a 4% frequency. However, vaccines with recombinant VP28 had the highest frequency of 12% among all treatments, with a survival rate between 50% and 75%.

WSSV vaccine production for Litopenaeus spp.
Penaeidae is a crustaceans family of great commercial value 106,107 . Among its different genera, Litopenaeus stands out as one of the most important shrimp species in the world industry [108][109][110] . Litopenaeus vannamei is among the principal species of this genus, commonly known as Pacific white shrimp111, the main farming species on the Ecuadorian coast 48 . However, this genus is prone to devastating diseases such as WSSV, which generate significant economic losses, and no commercial cure can eradicate the disease. Table 2 shows recent reports on conditions affecting the Litopenaeus genus, showing some updated general approaches to fighting them.
According to the research reviewed, vaccine manufacturing has been carried out ex-situ. Therefore, this techno-logy is still limited to the laboratory level. Further studies on production scale-up should be carried out to reduce costs, maintain product quality and develop in situ trials, allowing more accurate data to be generated during shrimp treatment.
Whispovirus vaccines aren't currently being commercialized at large-scale in the industry because of the high degree of variation in response to laboratory-tested vaccines and the high economic value of vaccine development 9,115 . Nevertheless, interest in controlling the devastating effects of the virus on Litopenaeus vannamei farms has led to increased interest in producing a vaccine that is efficient and affordable for field application.
The most efficient way to immunize Litopenaeus spp. with vaccines is by oral or infusion as it is not productive at the industrial level to apply it intramuscularly as this implies the application of the vaccine organism by the organism.
The vaccine industry and production is a complex activity with risks, which takes place in a harsh environment. Protocols for potential occupational hazards are necessary concerning contamination issues such as, product contamination, cross-contamination, amplification of contaminants, infection of workers and contamination of the environment 116 .
The animal vaccines currently available worldwide are developed by the veterinary pharmaceutical industry. Developing a vaccine requires an economic effort that takes years to perfect and guarantees its safety. Industrial development usually starts after laboratory testing that is based on solid academic research. A vaccine can only be made available to the veterinary community once the authorities have granted marketing approval, verifying its effects and potential harm 117 .
Industrial development must be seen in an economic context, which is not always the case in academic research so the use of reagents has large economic differences.
Farm Animals' vaccines are produced in large quantities at low cost, while vaccines for companion animals are produced in smaller quantities and sold at higher prices. It should also be taken into account that for-profit companies will generate the development of vaccines for higher incidence diseases or vaccines for high population species 118 .
In the case of shrimp, being a species of large-scale production generates interest in aquaculturists, and although Whispovirus is sporadic, it generates losses that affect shrimp production during these periods of appearance 42 . Figure 6 shows a production scheme for recombinant protein vaccines that could be used for further implementation in the industry. There is a small amount of commercialization of shrimp vaccines against WVVS. Yet, it is guessed that by having an efficient and replicable treatment in any environment, an industrial process could be implemented for its elaboration and oral administration.
According to figures 4 and 5 of the results obtained from the extracted articles, the production of vaccines with 2 genes has had a higher effectiveness rate. It confers more excellent protection to shrimps, being a process that can be used at the industrial level 61,83 .
The bacteria most commonly involved in the replication of recombinant proteins are Escherichia coli and Bacillus subtilis because of their more efficient replication, procurement and easy genetic manipulation [119][120][121] .

Conclusions
Antibiotics use on shrimp production cause: 1-potential adverse effect on human health 9,122 ,2-appearance of antibiotic-resistant strains 123,124 and 3-affections on shrimp larvae 125 . Contrarily, vaccine administration to control or lessen the incidence of vibriosis is an attractive choice nowadays.
Vaccination strategies against WSSV, such as inactivated viruses, subunit antigens, and DNA-based vaccines, have shown promise on a laboratory scale. However, drawbacks such as variable efficacy, high manufacturing cost, and limited field applicability need further investigation 126 .
A recent study describes a new attractive strategy based on RNAi technologies and polyanhydride nanopar-ticle-based delivery to develop a nanovaccine 108 . In aquaculture systems, the concept of RNAi-based vaccines has been advocated for several reasons: (a) RNAi functions as an antiviral immune response in shrimp; (b) it is pathogen-specific; and (c) it generates a long-term protective immune response.
On the other hand, another new technology combining vaccines with prebiotics has been shown to maximize the protective efficacy127-129 (Table 1). β-glucans, for example, is a joint prebiotic used in aquaculture and has long been used as an additive in the fish diet to improve the immune response enhancing the innate immune respon-se127, 130,131,132. Despite all these new alternatives to vaccine production and applications, more and more research, mainly on field trials, needs to be carried out to validate further and enhance the vaccine application effectiveness in shrimp.

Funding
This research received no external funding.

Institutional Review Board Statement
Not applicable.

Informed Consent Statement
Not applicable.

Acknowledgments
Authors thanks to Ing. Janyna Calderón Martinez and Ing. Christian Ortega for the critical reading of this manuscript. Their suggestions improved its content. Also, thanks to Ivan Andres Proaño and Björn Ludwig for their help in improving the manuscript's English graphics.

Conflicts of Interest
The authors declare no conflict of interest.