Alpacas and Human Interactions

Nanobodies.

One result of virus infection in animals is the production of antibodies by plasma B-cells. Antibodies are immune system proteins responsible for neutralising viruses by binding to them and preventing infection of further cells. Most mammals produce only one form of antibody that is made from a combination of heavy and light protein chains (see image). These form a 'Y'-shaped structure with the heavy chain proteins forming the backbone of the entire 'Y' and the light-chain proteins lining the arms. The backbone has relatively consistent structure but its few variants define the antibody's class: IgA, IgD, IgE, IgG, or IgM. It is the top of the 'Y'-shape that contains a region which tightly and specifically binds to an area on the antigen. Variations in this region allow for specific binding to a vast range of different antigen structures. Antibodies are large proteins of about of about 150 kDa and may be around 10 nm in size.

Diagram of a typical antibody and antigens

Llamas and alpacas differ, as in addition to the antibody structure described, they also possess a smaller variant (around 40% of the total antibodies) known as a single domain antibody [27]. This antibody form still has the ‘Y’ structure though the arms are shorter due to the absence of light-chain proteins - the variable regions are still located along the arms however. Separation and isolation of the arms from the stem yields the nanobody.

COVID-19

The Coronavirus family of viruses causes a wide range of illnesses in animals. These range from the common cold to Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS) in humans but can also cause diarrhoea, hepatitis, encephalomyelitis and respiratory illnesses in other species. A coronavirus infection causing diarrhoea in young camelid crias has also been identified [28 and 29]. These viruses can be transmitted between animals and people, a process known as zoonosis, so it is significant that wildlife can act as reservoirs of coronaviruses [31 and 32]. SARS-CoV-2, more widely known as COVID-19, is also a member of the Coronavirus group and likely originated from bats, possibly transmitting to humans through contact with pangolins, although this has yet to be proven.
To date, COVID-19 has proven transmissible to a variety of non-human animal species, eg. cats and dogs [59], great apes [60], large cats [61],[62], mink [63] and hippopotamus [64] (among others) resulting in differing degrees of disease severity. Recent research has shown that COVID-19 is unlikely to spill over into domestic livestock [58] which include cattle, sheep, goats, rabbits, horses and alpacas.

A critical element of the COVID-19 infection process is binding of the viral ‘spike’ glycoprotein to the cell receptor protein (angiotensin-converting enzyme 2 (ACE2)), a process that allows the virus particle to be taken into the cell. A nanobody, raised against the COVID-19 spike protein, strongly binds to it [33] thus preventing binding to the receptor and blocking cell entry. A variety of physical techniques (eg. Synchrotrons) are being used to determine the structure of nanobodies bound to the SARS-CoV-2 spike protein and determine the exact target area of the antigen.
The nanobodies are obtained by immunising an alpaca or llama with specific fragments of the virus' spike protein. After a number of days, blood is taken from the alpaca and the antibodies extracted. Their small size, solubility and stability make them attractive as candidate therapeutic agents. These properties give them the potential to be used similarly to a convalescent serum, effectively stopping progression of the disease and also developed into an inhaled treatment [35] since the nanobodies would be deposited directly into the lungs, the primary location of COVID-19 infection.
Findings published in an accelerated Nature paper [51] (7/6/2021) reveal that nanobody varieties raised in llamas, and mice engineered to produce nanobodies from other camelid species, remained effective even with receptor-binding domain antigenic drift, and recognised binding sites that are largely inaccessible to human antibodies. The work confirmed the efficacy of nanobodies and their potential in treatment of COVID-19 infection, especially in the light of emerging mutant strains. Recently, a paper has appeared [46] demonstrating that a further receptor, Neuropilin-1, which is abundant on the cells of the nasal cavity, significantly potentiates SARS-CoV-2 infectivity. The researchers showed that the effect is blocked by a monoclonal antibody against Neuropilin-1. Speculatively, this may be another suitable target for nanobodies.
Given the severity of the COVID-19 pandemic and the urgent need for innovative therapeutic solutions, there are several international scientific groups now researching this area using both alpacas and llamas as the source of nanobodies. A notable result (23/2/2021) is highlighted in "The Guardian", which reports the development of a electrochemical coronavirus test for Covid-19 using camelid nanobodies. This test is three times faster than lateral flow antigen tests and almost as accurate as PCR assays.

Nanobody-based Antivenoms for Snake Bites.

It is estimated that 5.4 million people are bitten each year by venomous snakes with 2.7 million affected by the toxin. Of these, around 110,000 victims die as the toxin may cause paralysis that can prevent breathing, bleeding disorders leading to a fatal haemorrhage, permanent kidney failure and other tissue damage. In addition, about three times this number require amputations or suffer other permanent disabilities [53].
The usual method of treatment is administration of an antivenom. This is produced by immunising horses or sheep with the venom of a particular snake or multiple toxins from a range of snakes and later extracting and purifying the resulting antibodies from the blood. When given to a patient, the specific antibodies bind to the venom or venom components in circulation and neutralise their activities.
An Australian innovation has been antivenom production from alpacas [54]. These were created for use against venoms from their local snakes, reputedly some of the most toxic in the world. This approach has also been taken by Peruvian investigators [55] using a llama for raising an antivenom for bites from the common lancehead (fer-de-lance) (Bothrops atrox), responsible for causing many fatalities in South America. In both cases, the camelid antivenoms had different efficacies to conventionally produced antivenoms but had comparable to superior activities against some venom components. The value of this approach was shown in the successful treatment of a dog that was bitten by a tiger snake [56]. The use of camelids for antitoxin production is likely to increase if only because nanobodies are able to bind to previously inaccessible antigen sites on the toxin components.

Since the discovery of camelid nanobodies, a large number of review papers have been published describing a wide variety of potential applications in the bio-medical fields, for examples [36, 37, 44, 45 and 48]. For full appreciation of the breadth of research, readers are recommended to consult these papers.

Further published advancements will be listed below:

Alpaca-derived single domain antibodies effectively neutralized typhoid toxin in vivo. Reported was a 100% survival rate of mice administered a lethal dose of the typhoid toxin [66].
The development of alpaca-derived multivalent nanobodies for the detection of the Chikungunya virus [70]. Chikungunya virus uses a mosquito vector for person to person transmission and is the causative agent of chikungunya fever. It is a re-emerging disease in Asia and although it is highly infectious, it has low mortality.
An alpaca antibody-based defense to protect plants from disease [72]. A protein from the targeted plant pathogen was innoculated into an alpaca, the resulting nanobody purified and the corresponding gene segment engineered into the experimantal plant’s own immune genes. This approach conferred strong resistance to the pathogen.
Construction of an alpaca immune antibody library for the selection of nanobodies against Drosophila melanogaster proteins [79]. Drosophila melanogaster is a model organism for studying developmental biology and human neural disorders. An immune VHH library against Drosophila embryo proteins was generated which allowed for highly efficient panning for nanobodies against proteins in Drosophila.

References.

Most of the literature below can be accessed by clicking on the highlighted link. Some links will access the appropriate web page from which the article can be downloaded but others will immediately start downloading the full reference.

27. Hamers-Casterman, C., Atarhouch, T., Muyldermans, S., Robinson, G., Hamers, C.,Songa, E.B., Bendahman, N., Hamers, R. (1993). Naturally occurring antibodies devoid of light chains. Nature, 363: 446–448.

28. Cebra, C.K., Mattson, D.E., Baker, R.J., Sonn, R.J., Dearing, P.L. (2003). Potential pathogens in feces from unweaned llamas and alpacas with diarrhea. J. Am. Vet. Med. Assoc. 223(12): 1806–1808.

29. Jin, L, Cebra, C.K., Baker, R.J., Mattson, D.E., Cohen, S.A., Alvarado, D.E. and Rohrmann, G.F. (2007). Analysis of the genome sequence of an alpaca coronavirus. Virol., 365(1): 198-203.

30. Vanlandschoot, P., Stortelers, C., Beirnaert, E., Ibañez, L.I., Schepens, B., Depla, E., Saelens, X. (2011). Nanobodies: new ammunition to battle viruses. Antivir. Res., 92: 389-407.

31. Poon, L.L.M., Chu, D.K.W., Chan, K.H., Wong, O.K., Ellis, T.M., Leung, Y.H.C., Lau, S.K.P., Woo, P.C.Y., Suen, K.Y., Yuen, K.Y., Guan, Y. and Peiris, J.S.M. (2005). Identification of a Novel Coronavirus in Bats. J. Virol., 79(4): 2001–2009.

32. Lam, T.T., Jia, N., Zhang, Y. et al. (2020). Identifying SARS-CoV-2-related coronaviruses in Malayan pangolins. Nature, 583: 282–285.

33. Hanke, L., Perez, L.V., Sheward, D.J., Das, H., Schulte, T., Moliner-Morro, A., Corcoran, M., Achour, A., Karlsson Hedestam, G.B. Hällberg, B.M., Murrell, B. and McInerney, G.M. (2020). An alpaca nanobody neutralizes SARS-CoV-2 by blocking receptor interaction. Posted 8th July 2020. doi: https://doi.org/10.1101/2020.06.02.130161.

35. Gai, J., Ma, L., Li, G., Zhu, M., Qiao, P., Li, X., Zhang, H., Zhang, Y., Chen, Y., Gong, R. and Wan, Y. (2020). A potent neutralizing nanobody against SARS-CoV-2 with inhaled delivery potential. BioRxiv Preprint server.

36. Jovčevska, I. and Muyldermans, S. (2020). The Therapeutic Potential of Nanobodies. BioDrugs, 34: 11-26.

37. Steeland, S., Vandenbroucke, R.E. and Libert, C. (2016). Nanobodies as therapeutics: big opportunities for small antibodies. Drug Disc. Today, 21(7): 1076-1113.

38. Lin, J., Gu, Y., Xu, Y., Yu, J., Tang, J., Wu, L., Zhou, Z., Chen, C., Liu, M., Chun, X., Liu, H., Nian, R., Song, H. and Zhang, J. (2020). Characterization and applications of nanobodies against Pseudomonas aeruginosa Exotoxin A selected from single alpaca B cells. Biotech. Biotechnol. Equip., 34(1): 1028–1037.

39. Chames, P. and Rothbauer, U. (2020). Special Issue: Nanobody. Antibodies 2020, 9(1), 6; https://doi.org/10.3390/antib9010006

40. Ji, L., Dong, C., Fan, R. and Qi, S. (2020). A high affinity nanobody against endothelin receptor type B: a new approach to the treatment of melanoma. Mol. Biol. Rep., 47, 2137-2147.

41. Xia, L., Teng, Q., Chen, Q. and Zhang, F. (2020). Preparation and Characterization of Anti-GPC3 Nanobody Against Hepatocellular Carcinoma. Int. J. Nanomed., 15: 2197-2205.

42. Dumoulin, M., Last, A.M., Desmyter, A., Decanniere, K., Canet, D., Larsson, G., Spencer, A., Archer, D.B., Sasse, J., Muyldermans, S., Wyns, L., Redfield, C., Matagne, A., Robinson, C.V. and Dobson, C.M. (2003). A camelid antibody fragment inhibits the formation of amyloid fibrils byhuman lysozyme. Nature, 424: 783-788.

43. He, Y., Ren, Y., Guo, B., Yang, Y., Ji, Y., Zhang, D., Wang, J., Wang, Y. and Wang, H. (2020). Development of a specific nanobody and its application in rapid and selective determination of Salmonella enteritidis in milk. Food Chem., 310, 25 April 2020, 125942.

44. Chames, P. and Rothbauer, U. (2020). Special Issue: Nanobody. Antibodies, 9(1): 6-9.

45. Schumacher, D., Helma, J., Schneider, A.F.L., Leonhardt, H. and Hackenberger, C.P.R. (2018). Nanobodies: Chemical Functionalization Strategies and Intracellular Applications. Angew. Chem. Int. Ed., 57: 2314-2333.

46. Cantuti-Castelvetri, L., Ojha, R., Pedro, L. D., Djannatian, M., Franz, J., Kuivanen, S., van der Meer, F., Kallio, K., Kaya, T., Anastasina, M., Smura, T., Levanov, L., Szirovicza, L., Tobi, A., Kallio-Kokko, H., Österlund, P., Joensuu, M., Meunier, F. A., Butcher, S. J., Winkler, M.S., Mollenhauer, B., Helenius, A., Gokce, O., Teesalu, T., Hepojoki, J., Vapalahti, O., Stadelmann, C., Balistreri, G. and Simons, M. (2020). Neuropilin-1 facilitates SARS-CoV-2 cell entry and infectivity. Sci., 20 Oct.

48. Yang E. Y., Shah K. (2020). Nanobodies: Next Generation of Cancer Diagnostics and Therapeutics. Front. Oncol., 23 July.

51. Xu, J., Xu, K., Jung, S. et al. (2021). Nanobodies from camelid mice and llamas neutralize SARS-CoV-2 variants. Nature. https://doi.org/10.1038/s41586-021-03676-z.

53. World Health Organisation (2021). Snakebite envenoming. Available online. Accessed 20/10/2021.

54. Padula, A. M. and Winkel, K.D. (2017). Antivenom production in the alpaca (Vicugna pacos): Monovalent and polyvalent antivenom neutralisation of lethal and procoagulant toxins in Australian elapid venoms. Small Ruminant Res., 149: 34-39.

55. Calderon, H.B., Coronel1, V.O.Y., Rey, O.A.C., Alave1, E.G.C., Duran, W.J.L., Rojas, C.P., Arevalo, H.M., Neyra, D.G., Pérez, M.G., Bonilla, C., Tintaya, B., Ricciardi, G., Smiejkowska, N., Romão, E., Vincke, C., Lévano, J., Celys, M., Lomonte, B. and Muyldermans, S. (2020). Development of Nanobodies Against Hemorrhagic and Myotoxic Components of Bothrops atrox Snake Venom. Front. Immunol., 11: 1-12.

56. Padula, A. M. and Winkel, K.D. (2016). Successful use of camelid (alpaca) antivenom to treat a potentially lethal tiger snake (Notechis scutatus) envenomation in a dog. Toxicon, 114: 59-64.

58. Bosco-Lauth, A.M., Walker, A., Guilbert, L., Porter, S., Hartwig, A., McVicker, E., Bielefeldt-Ohmann, H. and Bowen, R.A. (2021). Susceptibility of livestock to SARS-CoV-2 infectionEmerging Microbes and Infections, 10: 2199-2201.

59. Calvet, G.A., Pereira, S.A., Ogrzewalska, M., Pauvolid-Corrêa, A., Resende, P.C., Tassinari, W. de S., de Pina Costa,A., Keidel, L.A., et al. (2021) Investigation of SARS-CoV-2 infection in dogs and cats of humans diagnosed with COVID-19 in Rio de Janeiro, Brazil. PLoS ONE 16(4): e0250853. https://doi.org/10.1371/journal.pone.0250853

60. UNESCO (2021). Gorillas test positive to COVID-19: what it means for Great Apes. Web article, 12th January 2021.

61. France 24 report (2021). Lions at Singapore wildlife park infected with coronavirus. Web article, 10th November 2021.

62. Smithsonian’s National Zoo & Conservation Biology Institute (2021). Great Cats Tested Presumptive Positive For COVID-19 at the Smithsonian's National Zoo. Web article, 17th September 2021.

63. Chaintoutis SC, Thomou Z, Mouchtaropoulou E, Tsiolas G, Chassalevris T, Stylianaki I, et al. (2021). Outbreaks of SARS-CoV-2 in naturally infected mink farms: Impact, transmission dynamics, genetic patterns, and environmental contamination. PLoS Pathog., 17(9): e1009883.

64. BBC News (2021). Belgian zoo hippos test positive for Covid. Web article, 4th December 2021.

66. Dulal, H.P., Vance, D.J., Neupane, D.P., Chen, X., Tremblay, J.M., Shoemaker, C.B., Mantis, N.J. and Song, J. (2022). Neutralization of typhoid toxin by alpaca-derived, single-domain antibodies targeting the PltB and CdtB subunits. Infect. Immun., 90(2): Feb.

70. Li, Q., Zhang, F., Lu, Y., Hu, H., Wang, J., Guo, C., Deng, Q., Liao, C., Wu, Q., Hu, T., Chen, Z. and Lu, J. (2022). Highly Potent Multi-Valent Nanobodies Against Chikungunya with VHH Screened from Alpaca Naïve Phage Display Library. DOI: Research Square pre-publication.

71. Panteleev, P.V., Safronova, V.N., Kruglikov, R.N., Bolosov, I.A., Bogdanov, I.V. and Ovchinnikova, T.V. (2022). A Novel Proline-Rich Cathelicidin from the Alpaca Vicugna pacos with Potency to Combat Antibiotic-Resistant Bacteria: Mechanism of Action and the Functional Role of the C-Terminal Region. Membranes, 12: 515-533.

72. Stokstad, E. (2023). Antibody-based defense may protect plants from disease. Sci., 379(6635): 867..

79. Qiu, J., Li, J., Zhang, Z., Dong, S., Ling, X., Fang, Z., Ling, Q. and Huang, Z. (2023). Construction of an alpaca immune antibody library for the selection of nanobodies against Drosophila melanogaster proteins. Sci., 379(6635): 867.Front. Bioeng. Biotechnol. 11: 1207048.